The spatio-temporal distribution of aromatase cytochrome in ovary throughout the canine oestrous cycle.

CONTEXT: New animal welfare legislation and ethical guidelines encourage alternative approaches for canine contraception, instead of surgical gonadectomy which is considered invasive and unjustified in healthy dogs. AIMS: Reversible contraception might be achieved by inhibition of aromatase (CYP19), an enzyme catalysing the conversion of androgens to oestrogens. This study provides insights into the spatio-temporal expression and distribution of aromatase in canine ovarian tissue. METHODS: Ovarian tissue was collected from 39 healthy and sexually mature bitches during different stages of the oestrous cycle: pro-oestrus (n =8), oestrus (n =12), dioestrus (n =9) (luteal phase) and anoestrus (n =10). Localisation of cytochrome P450 aromatase was determined by immunohistochemistry. KEY RESULTS: Aromatase activity in the dog is high during pro-oestrus, ovulation and early dioestrus. Comparing types of follicles and corpora lutea, the highest aromatase abundance was found in antral follicles and luteinising follicles, whereas corpora lutea and early antral follicles showed an intermediate presence of the enzyme. Interesting was the high abundance of aromatase in luteinising theca interna cells, prevailing over granulosa cells. CONCLUSIONS AND IMPLICATIONS: Understanding of cells involved in oestradiol production is important for targeted inhibition of oestradiol synthesis, possibly offering an approach for contraception and suppression of oestrus.

Administration of aromatase inhibitor MPV-2213ad to blue fox vixens (Vulpes lagopus) as a model for contraception in female dogs.

The interest in non-surgical approaches to contraception and fertility control in female dogs has increased in recent years. In this study the effect of an aromatase inhibitor (finrozole) was evaluated in fur production animals, farmed blue fox vixens, as a model for contraception in bitches. A total of 80 vixens were divided into 4 groups, receiving orally placebo (A) or finrozole 0.5 mg/kg (B), 3.5 mg/kg (C) or 24.5 mg/kg (D) for 21 consecutive days beginning in the pre-ovulatory period of heat. Monitoring of the vixens included clinical signs of heat, measurement of vaginal electrical resistance (VER) as well as oestradiol and progesterone concentrations in plasma. The approximate relation of the start of treatment to ovulation varied from 11 days before to one day after ovulation provided that the LH peak occurred 0.5-2 days before the VER peak and ovulation was then estimated to occur 2 days after the LH peak. Seventy vixens were artificially inseminated within 8 h after a 50 Ω decline in vaginal electrical resistance was detected. Ten vixens were not inseminated. Pregnancy was confirmed by transabdominal ultrasound examination and birth of cubs was recorded. The pregnancy rates in the groups were 89.5% (A), 81.3% (B), 55.6% (C) and 52.9% (D). The average number of live born pups in the four groups was 9.4 (A), 7.0 (B), 5.8 (C), and 3.8 (D), respectively. No deleterious effects (for instance malformations) of finrozole on pups could be verified. The administration of finrozole did not have a significant effect on oestradiol parameters and VER values in vixens. Progesterone values were significantly higher in treatment groups compared with the placebo group. The results indicate that pregnancy could be avoided by finrozole provided that doses of ≥3.5 mg/kg were used and the treatment was initiated at least four days before the day of artificial insemination. This corresponds with two to six days before ovulation provided that the LH peak occurred 0.5-2 days before the VER peak and that ovulation then occurred in average 2 days after the LH peak.

Strategies for a direct characterization of phosphoproteins on hydroxyapatite surfaces.

We show in this work how systems formed by phosphoproteins on calcium phosphate surfaces can be directly characterized, in real time, in liquid medium, without the need for elution or labeling. Specifically, we show how this is possible by applying three different techniques: ellipsometry, quartz crystal microbalance with dissipation, and atomic force microscopy-based friction force spectroscopy. We apply these techniques to study two different model systems, i.e. those formed upon the adsorption of two model phosphoproteins (κ- and ß-casein) on hydroxyapatite (HA) surfaces. Information on the kinetics of adsorption, surface excess, viscoelasticity, water content, thickness of the layers, and protein-surface interaction is provided. Results indicate that both phosphoproteins form homogeneous elastic highly hydrated monolayers on the HA surfaces, the strength of ß-casein layers being higher by approximately a factor of 4. Based on the experimental results, models for the conformation of κ- and ß-casein molecules adsorbed on HA surfaces are proposed.

Nanowear of salivary films vs. substratum wettability.

The pellicle serves as a multifunctional protective layer, providing, e.g., lubrication and remineralization and also acting as a diffusion barrier. In addition, since the formation of the pellicle precedes the adhesion of micro-organisms, it is also important as a conditioning film. We present a novel approach to study the influence of the water wettability of solid surfaces on the strength of adsorbed salivary films. It is based on studying the wear resistance of the films with an atomic force microscope operated in the friction force spectroscopy mode. This methodology provides the strength of the films in terms of the forces needed for breaking and removing them. Our results indicate that these forces are highly dependent on the water wettability of the underlying substrata, decreasing with increasing hydrophobicity. Thus, this study provides valuable information for the design of materials exposed in the oral cavity, i.e., materials that will minimize plaque formation and be easy to clean.

Improving teamwork between students from two professional programmes in dental education.

In Sweden, the National Board of Health and Welfare forecasts a decrease in dentists with 26% and an increase in dental hygienists with 47% until the year of 2023. This, together with changes in both epidemiology, especially of dental caries, and political priorities, calls for an effective and well-developed cooperation between dentists and dental hygienists in future dentistry. Hence, the aim of this project was to investigate whether highlighting teamwork during the undergraduate studies of dental students and dental hygiene students could improve the students' holistic view on patients as well as their knowledge of and insight into each other's future professions. Thirty-four dental students and 24 dental hygiene students participated in the study. At the beginning of their final year in undergraduate education, a questionnaire testing the level of knowledge of the dental hygienists' clinical competences was completed by both groups of students. In addition, activities intending to improve teamwork quality included the following: (i) a seminar with a dentist representing the Public Dental Health Services in Sweden, (ii) dental students as supervisors for dental hygiene students, (iii) planning and treatment for shared patients and (iv) students' presentations of the treatments and their outcomes at a final seminar. The project was ended by the students answering the above-mentioned questionnaire for the second time, followed by an evaluation of the different activities included in the study. The knowledge of dental hygienists' competences showed higher scores in almost all questions. Both groups of students considered the following aspects important: seminars with external participants, dental students acting as supervisors and planning and treating shared patients. By initiating and encouraging teamwork between dental students and dental hygiene students, it is possible to increase knowledge on dental hygienists' competence and also to develop and strengthen a holistic view on patients and dental work, thereby preparing both groups of students for their professional life.

The composition of enamel salivary films is different from the ones formed on dental materials.

This study utilized two-dimensional gel electrophoresis (2DE) to illustrate the compositional differences between in vitro salivary conditioning films (denoted pellicles) formed on human enamel as well as on the dental materials titanium and poly(methyl methacrylate). The salivary pellicles were formed by immersing each surface in individual tubes containing small volumes of freshly collected whole saliva. Saliva remaining in the tubes after the pellicle formation for 2 h was visualized by means of 2DE and silver staining. The results showed that the protein patterns in 2DE of the liquid phase of saliva left after the exposure to the respective surfaces, regarding proteins <100 kDa in size, were different depending on the surface used. Several protein groups and/or individual proteins were shown to be distinct for each surface used.

Film-forming properties and viscosities of saliva substitutes and human whole saliva.

Hypo-salivation, related to medical remedies, is an increasing clinical problem. Studies report a weak correlation between subjective mouth dryness and objective sialometry. This indicates that both quantity and quality of saliva are important for the surface-associated functions of saliva, such as lubrication and hydration, to be expressed. Film-forming properties and viscosities of three saliva substitutes were compared to human saliva. Adsorption to surfaces was measured by ellipsometry, infrared spectroscopy and drop-volume technique. Viscosity measurements were carried out using an oscillating rheometer. Saliva, with the lowest viscosity value and the highest protein content, presented superior film retention on both hydrophilic and hydrophobic surfaces. The carboxymethylcellulose-based MAS 84 showed intermediate values of viscosity, poorest ability to reduce surface tension, and negligible film-forming capacity. The porcine mucin-based Saliva Orthana showed about twice the viscosity of saliva and film-forming capability on preferably hydrophobic substrates. Salinum, a linseed extract, possessed the highest viscosity value and an initial surface tension close to that of saliva. The film retention on hydrophilic surfaces was not as effective as for saliva. The results indicate that the film-forming capacity of saliva substitutes is a property also to be considered in the exploration of clinically effective artificial salivas.

Bacteria on stumps of amputees and the effect of antiseptics.

Amputees get stump infections usually from the natural inhabitants of the healthy skin and probably due to the unnatural environment of tight fitting sockets. The aim of the present study was to investigate the natural stump bacteria and the effect of antiseptics as well as the amputees' evaluation of such treatment. Fifteen amputees using their prostheses all day were investigated. Bacterial samplings were taken by swab technique with respect to bacteria and fungi from the stumps in the morning before prosthetic application and in the evening after a whole day's prosthetic use without antiseptic cleaning; after antiseptic cleaning with a combination of Isopropanol 45%. N-propanol 30% and N-cetyl-pyridiniumchloride 0.2% for one day: after fourteen days continuous use. The patients were asked if they liked the antiseptic and if they would like to continue to use it. Two patients did not submit bacteriological samples after the cleaning period. Before cleaning S. epidermidis, S. aureus and alpha-hemolytic streptococci were commonly found. In two instances gram negative rods were found. After the cleaning period there was a reduction of bacteria in 11 out of 13 patients. All patients liked the antiseptic and the simplicity by which the stumps and the sockets could be kept clean. The authors feel that the use of antiseptics to increase stump and socket hygiene is justified.

Comparison of CAD-CAM and hand made sockets for PTB prostheses.

The aim of the present study was to compare sockets for below-knee (BK) prostheses made by Computer Aided Design-Computer Aided Manufacture (CAD-CAM) to those made by hand. The patients in the study were provided with two prostheses each, which apart from the sockets, were identical. One socket was made by the CAD-CAM technique developed at the Bioengineering Centre, Roehampton, University College London and one was made by hand at the OT-Centre, Stockholm, Sweden. The results were based on investigation of eight unilateral below-knee amputees evaluating their own sockets by Visual Analogous Scale with respect to comfort, pressure, and pain. The sockets were evaluated on seven occasions, at two tests, on delivery, after use every second day for six days and every second week for two weeks. All CAD-CAM sockets except one had to be changed once as compared to the hand made of which only two had to be changed. As to comfort it could not be demonstrated that there was any significant difference between the two types of sockets and both types were well accepted by all patients. Differences in pressure and pain were rarely reported. There were obvious differences between the two types of socket with respect to height, width, and inner surface configuration. The authors feel that CAD-CAM will in the near future be an excellent tool for design and manufacture of prosthetic sockets.

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography.

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.

Cyproterone acetate prevents translocation of the androgen receptor in the rat prostate.

The translocation of the androgen receptor in prostatic tissue has been studied under the influence of different ligands (testosterone, methyltrienolone and cyproterone acetate) in vivo and in vitro. Nuclear and cytoplasmic androgen receptors were estimated using an exchange assay with [3H]methyltrienolone ( [3H]R1881) 1 h and 16 h after injection in castrated rats of either 100 micrograms testosterone (T), 10 mg cyproterone acetate (CA) or the combination of T and CA. Within 1 h after T administration, nuclear receptor levels increased with a concomitant depletion of cytosol receptors. In the CA-treated rats nuclear receptor levels were not different from those of control castrated animals and there was no depletion of cytosol receptors. The combined treatment of T and CA resulted in a partial depletion of cytosol receptors and a simultaneous increase of nuclear receptors. The absence of an increase in nuclear androgen receptors in CA-treated animals cannot be explained by a delay in translocation, because even 16 h after CA injection, only a very small number of nuclear receptors were detectable. Incubation of minced prostatic tissue with [3H]CA or [3H]R 1881 resulted in receptor translocation only in the R1881 incubations and confirmed the in vivo results. Competition studies with different steroids and cytosol receptor (non-activated, 8S form in low salt gradient) or nuclear receptor (activated 3.6S form in high salt gradient) of prostatic tissue show that CA can compete with R1881 for specific androgen-binding sites with a similar relative binding affinity for both receptor preparations. The present results provide evidence that CA prevents translocation of the androgen receptor to the nucleus, although CA can be bound with similar affinities to the nuclear receptor and the cytoplasmic receptor. We propose that the anti-androgenic action of CA involves an inhibition of receptor translocation.

The effects of short-term culture and perifusion on LH-dependent steroidogenesis in isolated rat Leydig cells.

Tumour Leydig cells and normal mature Leydig cells lost their steroidogenic response (pregnenolone and testosterone secretion) to LH after 24 h of culture. Immature cells showed a 2-fold increase in the basal pregnenolone secretion and no change in the LH-dependent pregnenolone secretion after 24 h of culture, whereas the LH-dependent steroidogenesis decreased after 48 h. None of the 3 cell preparations showed morphological signs of degeneration during a culture period of more than 7 days. Histochemical 3 beta-hydroxysteroid dehydrogenase activity in isolated immature Leydig cells disappeared during the first 2 days of the culture period and re-appeared after 5--7 days. Testosterone production by mature Leydig cells decreased during the first hours after exposure to LH, whereas pregnenolone secretion remained constant. From these results it was concluded that Leydig cells attached to plastic can be used for investigation of acute actions of LH on steroidogenesis. A perifusion technique for cells attached to plastic was developed and was applied to the kinetics of LH action on steroidogenesis in tumour and immature Leydig cells. The first stimulation of pregnenolone secretion occurred within 5 min, but a full stimulation was only obtained after 20--30 min. This was followed by a gradual decrease in the stimulated steroid secretion to approximately 50% after 60 min.

The mechanism of action of lutropin on regulator protein(s) involved in Leydig-cell steroidogenesis.

The dependence on lutropin of the synthesis of a proposed short-half-life protein regulator involved in Leydig-cell steroidogenesis was investigated. This was carried out by determining the effect of the protein-synthesis inhibitor cycloheximide, added before and during incubations with lutropin (and/or dibutyryl cyclic AMP), on the rate of testosterone production in suspensions of purified Leydig cells from adult rat testes. The Leydig cells were preincubated in Eagle's medium for 2.5h followed by 30min incubation with and without cycloheximide. The inhibitor was removed by washing the cells and then lutropin was added and testosterone concentrations were determined after incubation of the cells at 32 degrees C. No significant effect of cycloheximide pretreatment on lutropin-stimulated steroidogenesis was found during 60min incubation. This was in contrast with the complete inhibiting effect of cycloheximide when it was added with the lutropin. The pretreatment experiments with cycloheximide were repeated in the presence of dibutyryl cyclic AMP and elipten phosphate (to inhibit cholesterol side-chain cleavage) followed by incubation with lutropin. After 5, 10, 20 and 60min of incubation, testosterone concentrations were 61+/-3, 46+/-3, 27+/-4 and 18+/-4% lower than in the cells pretreated without cycloheximide respectively (means+/-s.e.m., n=4-6). In the cells not pretreated with cycloheximide and in the absence of lutropin, testosterone production increased from 1.36+/-0.5 to 36.5+/-1.0ng/10(6) cells during 20min of incubation, after which no further increase occurred. Pretreatment of the cells with cycloheximide decreased these testosterone concentrations by 65, 46, 42 and 36% in the 5, 10, 20 and 60min incubations respectively (mean values, n=2-4). It is apparent from these results that inhibition of steroidogenesis only occurs if protein synthesis is inhibited in the presence of lutropin or cyclic AMP. A new hypothesis is put forward to explain these findings: it is proposed that lutropin affects the stability of a precursor of a regulator protein by converting it from a stable (inactive) to an unstable (active) form with a short half-life.

A Leydig cell tumour: a model for the study of lutropin action.

The properties of cells isolated from a Leydig cell tumour have been compared with normal rat testis Leydig cells. These cells were found to be similar in the following respects: 1. Lutropin-stimulated cyclic AMP and testosterone production. 2. Lutropin-activated protein kinase activity followed by phosphorylation of endogenous proteins of mol. wts. 57,000and 14,000. 3. Parallel lutropin dose vs. response curves for phosphorylation of the endogenous proteins and for testosterone production. 4. Two forms of isoenzyme, cyclic AMP dependent protein kinase, present. They differed mainly with respect to the lutropin-stimulated testosterone production, which was much lower in the tumour cells compared with the normal adult testis Leydig cells (4.6 +/- 1.1 and 114 +/- 16 ng testosterone/10(6) cells per 2 h, respectively). However, the lutropin-stimulated steroid production in the tumour cells was quantitatively comparable with the normal rat Leydig cell when the metabolism of pregnenolone in intact cells and mitochondria was inhibited by addition of SU-10603 and/or cyanoketone. It is concluded that the Leydig cell tumour used in this study can be used to investigate certain aspects of lutropin action where large quantities of cells are required.