Digital holographic microscopy for the evaluation of human sperm structure.

The morphology of the sperm head has often been correlated with the outcome of in vitro fertilization, and has been shown to be the sole parameter in semen of value in predicting the success of intracytoplasmic sperm injection and intracytoplasmic morphologically selected sperm injection. In this paper, we have studied whether digital holographic microscopy (DHM) may be useful to obtain quantitative data on human sperm head structure and compared this technique with high-power digitally enhanced Nomarski optics. The main advantage of digital holography is that high-resolution three-dimensional quantitative sample imaging may be automatically produced by numerical refocusing of a two-dimensional image at different object planes without any mechanical scanning. We show that DHM generates useful information on the dimensions and structure of human sperm, not revealed by conventional phase-contrast microscopy, in particular the volume of vacuoles, and suggest its use as an additional prognostic tool in assisted reproduction technology.

Clinical characteristics and incidence of first fracture in a consecutive sample of post-menopausal women attending osteoporosis centers: The PROTEO-1 study.

BACKGROUND: Osteoporosis is a highly prevalent disease and fractures are a major cause of disability and morbidity. AIM: The purpose of this study was to characterize post-menopausal women attending osteoporosis centers in Italy, to evaluate physician management, and to determine the incidence of first osteoporotic fracture. SUBJECTS AND METHODS: PROTEO-1 was an observational longitudinal study with a 12-month follow-up. Data were collected from women attending osteoporosis centers. Women without prevalent fracture were eligible to enter the 1-yr follow-up phase: the clinical approach to patients according to their fracture risk profile and the incidence of fracture were recorded. RESULTS: 4269 patients were enrolled in 80 centers in the cross-sectional phase; 34.2% had an osteoporotic fracture at baseline. Patients with prevalent fractures were older and more likely to be treated compared with non-fractured patients. The incidence of vertebral or hip fracture after 1 yr was 3.84%, regardless of the calculated risk factor profile, and was significantly higher in patients with back pain at baseline (4.2%) compared with those without back pain (2.2%; p=0.023). Generally, physicians prescribed more blood exams and drugs to patients at higher risk of fracture. Among fractured patients only 24% were properly treated; the rate of non-responders to treatment was about 4%. CONCLUSIONS: In a large, unselected sample of post-menopausal women attending osteoporosis centers, those without previous fracture were at substantial risk of future fracture, regardless of their theoretical low 10-yr fracture risk. The presence of back pain in women without previous fracture warrants close attention.

Transcription factor expression, RNA synthesis and NADPH-diaphorase across the rat brain and exposure to spatial novelty.

The molecular hypothesis of learning and memory processes is based on changes in synaptic weights in neural networks. Aim of this study was to map neural traces of exposure to a spatial novelty were mapped by (i) the transcription factors (TFs) c-fos, c-jun and jun-B using Northern blot and immunocytochemistry (ICC), (ii) RNA synthesis by (3)H-uridine autoradiography and RNA level, (iii) NADPH-diaphorase (NADPH-d) expression by histochemistry. Thus, adult male albino rats were exposed to a Làt-maze and sacrificed at different times. Non-exposed rats served as controls. The latter showed a low constitutive expression of TF, RNA synthesis and NADPH-d across the brain. Northern blots showed a three-fold increase in TFs in exposed versus non-exposed rats in the cerebral cortex. ICC showed in exposed rats several TFs positive cells in the granular and pyramidal layers of the hippocampus and later in all layers of the somatosensory cortex, in the granular layer of the cerebellar cortex. The TF-positivity was stronger in rats exposed for the first time, and was time and NMDA-dependent. Autoradiography for RNA synthesis showed positive cells in the ependyma, hippocampus and cerebellum 6h after testing, and in the somatosensory cortex 24h later. In addition, exposure to novelty induced NADPH-d in the dorsal hippocampus, the caudate-putamen, all the layers of the somatosensory cortex. and the cerebellum. The positivity was absent immediately after exposure, appeared within 2h and disappeared 24h later. A strong neuronal discharge by the convulsant pentylenetetrazol, strongly induced TFs but not din not affect NADPH-d 2h later. Thus, data suggest that the processing of spatial and emotional components of experience activates neural networks across different organization levels of the CNS.

Validity and reliability of the Italian version of the ECOS-16 questionnaire in postmenopausal women with prevalent vertebral fractures due to osteoporosis.

OBJECTIVE: To investigate the reliability and validity of the Italian version of ECOS-16 (Assessment of health related quality of life in osteoporosis) in comparison to other questionnaires in postmenopausal women with osteoporosis. METHODS: A cross-sectional multicentre study was carried out among postmenopausal women with osteoporosis who were attending primary care centres and hospital outpatient clinics. The patient group included 234 females (mean age 69 years, range 48-89) who presented vertebral fractures due to osteoporosis. The control group consisted of 244 asymptomatic osteoporotic subjects matched for age with the patient group. The psychometric properties of the questionnaires were evaluated in terms of feasibility, validity (construct validity and discriminant validity) and internal consistency. Test-retest reliability was analysed for 196 outpatients who reported that their general health status due to osteoporosis had not changed after one week. In all patients the ECOS-16, the SF-36 (Medical Outcomes Study Short Form-36), EUROQoL (EQ-5D), mini-OQLQ (mini-Osteoporosis Quality of Life Questionnaire), and RMDQ (Roland-Morris Disability Questionnaire) were administered, and all clinical variables and sociodemographic variables were taken into account. Construct and discriminant validity were assessed by Spearman's correlations, the Wilcoxon rank sum test, the Kruskal Wallis test and by receiver operating characteristic (ROC) curves. Internal consistency was evaluated using Cronbach's alpha and the test-retest reliability was evaluated by intra-class correlation coefficients (ICCs). RESULTS: 96.9% of the patients answered all items of the ECOS-16 questionnaires. The mean administration time was 10 minutes. Factor analysis yielded two factors that accounted for 88.4% of the explained variance in the ECOS-16 questionnaire. The first factor was the ECOS-16 Physical Component Score (PCS) (45.9% of the explained variance) and the second factor was the ECOS-16 Mental Component Score (MCS) (42.4% of the explained variance). The inter-item correlation between the two factors was 0.48. Significant correlations were found between the scores of similar domains or subscales of the ECOS-16 and SF-36, EQ-5D and mini-OQLQ, supporting the concept of convergent construct validity. The total ECOS-16 score progressively increased with the number of prevelant vertebral fractures (p<0.001) and the effect of the first fracture was already statistically significant (p<0.01). On ROC curve analysis the total ECOS-16 score showed the highest performance among the different questionnaires in discriminating between patients with vertebral fractures and controls with no fractures. In the reliability study, internal consistency within the domain of ECOS-16 was generally good, with Chronbach's alpha values ranging from 0.81 to 0.89. Test-retest reliability was 0.87 for the total ECOS-16 score. CONCLUSION: The Italian version of the ECOS-16 questionnaire was demonstrated to have good psychometric properties and could offer a useful tool in research and routine clinical practice to evaluate HRQoL in post-menopausal women with osteoporosis. A full validation of the psychometric properties will require data on its sensitivity to change.

Cell biology of the harderian gland.

The harderian gland is an orbital gland of the majority of land vertebrates. It is the only orbital gland in anuran amphibians since the lacrimal gland develops later during phylogenesis in some reptilian species. Perhaps because it is not found in man, little interest was paid to this gland until about four decades ago. In recent years, however, the scientific community has shown new interest in analyzing the ontogenetic and morphofunctional aspects of the harderian gland, particularly in rodents, which are the preferred experimental model for physiologists and pathologists. One of the main characteristics of the gland is the extreme variety not only in its morphology, but also in its biochemical properties. This most likely reflects the versatility of functions related to different adaptations of the species considered. The complexity of the harderian gland is further shown in its control by many exogenous and endogenous factors, which vary from species to species. The information gained so far points to the following functions for the gland: (1) lubrication of the eye and nictitating membrane, (2) a site of immune response, particularly in birds, (3) a source of pheromones, (4) a source of saliva in some chelonians, (5) osmoregulation in some reptiles, (6) photoreception in rodents, (7) thermoregulation in some rodents, and (8) a source of growth factors.

Does testing for prostate-specific antigen contribute to declining prostate cancer mortality? Estimating the broader economic influences on aggregate prostate cancer mortality rates.

Key variables associated with prostate cancer mortality were examined using Canadian province level time-series data over the period 1979-1999 in a multiple regression framework. The key variables driving the prostate cancer mortality rate are per capita income, per capita number of family physicians, rate of prostate cancer incidence, real per capita total health spending, and a time trend. As well, provincial dummy variables show that regional differences exist with higher rates of mortality in eastern Canada. Econometric results show a positive and significant relationship between incidence and mortality, suggesting that the increased amount of prostate-specific antigen (PSA) testing may not have been responsible for mortality declines. However, there is also a downward trend in mortality from prostate cancer when all other factors are controlled for that can be attributed partly to the onset of PSA testing given that the benefits should emerge over time. A 1% increase in incidence is associated with a 0.2% increase in mortality. A 1% increase in real per capita income is associated with a 0.5% increase in mortality when real per capita income is below Canadian dollars 20,054. A 1% increase in the per capita number of family physicians reduces the mortality rate by 0.5%. A 1% increase in real per capita total health expenditures is associated with a 0.7% decline in the mortality rate. Finally, by 1999 there were 4.74 fewer deaths per 100,000 population due to the effect of time after controlling for all other factors--a decrease of approx. 15%.

A gonadotropin-releasing hormone (GnRH) antagonist decreases androgen production and spermatogonial multiplication in frog (Rana esculenta): indirect evidence for the existence of GnRH or GnRH-like material receptors in the hypophysis and testis.

The effects of a GnRH antagonist (GnRHA) on GnRH agonist (GnRH*)-induced androgen production and spermatogonial multiplication were studied in the frog, Rana esculenta, in vivo and in vitro. Intact and hypophysectomized (PDX) animals were kept at 22 +/- 2 C and treated with GnRH (45 ng/g BW) and GnRH* plus 1X and 10X concentrations of GnRHA on alternate days for 2 weeks. Androgen concentration in GnRH* plus GnRHA-treated animals decreased in the testis by about 50% with the 10X dose whereas the increase obtained in GnRH*-treated PDX group was completely abolished with the 1X dose. Histological sections were evaluated with respect of the mitotic index (MI) of the primary spermatogonia. Both GnRHA-treated intact and PDX frogs showed a dose-dependent MI decrease which reached 59% and 57% of control, respectively. In vitro incubations were carried out on testis halves at 15 C for 0, 2, 4, 6, and 8 h with the addition of 1 microgram GnRH* and 1 microgram GnRH* plus 1 or 10 micrograms GnRHA. The stimulatory effect of GnRH* and the inhibitory effect of GnRHA were apparent within 2 h. The basal mitogenic activity was affected by antagonist treatment and the inhibitory effect on the MI was evident within 2-4 h in the 10X-treated groups or within 6-8 h in the 1X treated groups. Since GnRH* and GnRHA bind to the same receptor these data strongly indicate that the effects of putative GnRH-like materials in the frog, Rana esculenta, are mediated throughout stereospecific recognition sites in both pituitary and testis.

In vivo and in vitro stimulatory effect of a gonadotropin-releasing hormone analog (HOE 766) on spermatogonial multiplication in the frog, Rana esculenta.

The effects of a GnRH analog (GnRHA), D-Ser-t-Bu6,desGly-NH2(10) (HOE 766) on spermatogenesis were analyzed in the frog, Rana esculenta. Intact animals caught at two different periods of the year (January and March) were treated with HOE 766 (GnRHA, 45 ng/g BW) at low (4 +/- 1 C) and high (22 +/- 2 C) temperatures. Hypophysectomized frogs were used also and, in addition to GnRHA, these animals were treated with crude pars distalis homogenate. In vitro incubations were carried out at 15 C, for 0, 6, and 24 h with the addition of 1 microgram GnRHA. Half of each testis was used as the untreated control. Histological sections of the testes were analyzed for the evaluation of the mitotic index of the primary spermatogonia. Intact March animals had mitotic indices higher than January animals. GnRHA treatment elicited an increase of the mitotic index in both intact and hypophysectomized animals. High temperature potentiated the GnRHA effect while low temperature favored pars distalis treatment. In conclusion, the present results are consistent with the fact that in the frog, R. esculenta, the magnitude of spermatogonial proliferation is temperature dependent, and for the first time it is shown that GnRH-like substances have direct stimulatory effect on the mitotic activity of the primary spermatogonia in a vertebrate.

Androgen receptor in the Harderian gland of Rana esculenta.

An androgen receptor has been identified in the cytosolic and nuclear extracts of the Harderian gland of the frog, Rana esculenta. A single class of high-affinity binding sites was found: Kd = 1.9 +/- 1.3 (S.D.) nmol/l (n = 26) for the cytosolic extract and Kd = 0.9 +/- 0.8 nmol/l (n = 15) for the nuclear extract. The presence of binding activity in both nuclear and cytosolic extracts and the low rate of ligand-receptor dissociation are characteristics that distinguish this receptor from a steroid-binding protein. The Kd did not show any sex difference and did not exhibit any secretory activity-related change. Binding in both cytosolic and nuclear extracts was specific for androgens (testosterone = 5 alpha-dihydrotestosterone); oestradiol-17 beta showed a 30% cross-reaction; moreover, specific binding of [3H]oestradiol-17 beta was not detectable. The binding capacity of the Harderian gland increased progressively in both fractions from October to December, reaching a peak in May, and decreased suddenly during July to August. The lack of any morphological sex-related difference in the Harderian gland of the green frog might be accounted for by the high amount of circulating androgens as well as a similar concentration of androgen receptor in both sexes.

Stimulatory effect of a GnRH agonist (buserelin) in in vitro and in vivo testosterone production by the frog (Rana esculenta) testis.

The summary testicular effects of an agonistic analogue of gonadotropin-releasing hormone (buserelin, GnRHa) have been studied in vitro and in vivo in the frog, Rana esculenta. During 3 h incubation GnRHa (8 X 10(-7) M) potentiated pituitary factors in stimulating testosterone production by minced testes in vitro. After 6 h of incubation 8 X 10(-7) M GnRHa stimulated maximal testosterone output. Testes of 10-day hypophysectomized animals did not show any GnRHa effect in vitro. In vivo, a direct effect of GnRHa on testicular testosterone production was demonstrated in hypophysectomized animals, although this effect was temperature-dependent, requiring the frog to be maintained at a high temperature (24 degrees C). No effect of GnRHa was detectable in frogs kept at a low temperature (4 degrees C).

Testosterone induction of poly(A)(+)-RNA synthesis and [35S]methionine incorporation into proteins of Rana esculenta Harderian gland.

The role of androgens in the cyclic secretory activity of the Rana esculenta Harderian gland (HG) was studied. Total RNA showed a dramatic increase in October and May when the nuclear androgen receptors peak. During the resumption of the secretory activity a gradual increase of poly(A)(+)-RNA was detected; during the enhancement phase (May) a peak of the poly(A)(+)-RNA fraction was found. In in vitro experiments testosterone increased the incorporation of [3H]uridine into the poly(A)(+)-RNA fraction and also that of [35S]methionine into a newly synthesized protein fraction (100 kDa). The latter effect is prevented by the exposure of the cells to the antiandrogen, cyproterone acetate (CPA). These findings reveal that, besides hamsters, the HG is a target for androgens in the frog.

Evidence on the determinants of Canadian provincial government health expenditures: 1965-1991.

Real per capital provincial government expenditures on health care over the period 1965-1991 are examined using pooled time-series cross-section regression analysis: Key determinants of real per capita provincial government expenditures on health care over the period 1965-1991 are real provincial per capita income, the proportion of the provincial population over age 65 and real provincial per capita federal transfer revenues. Established program financing had a negative and significant impact on real per capita provincial government health expenditures in Newfoundland and Quebec. An income elasticity of 0.77 implies that health care is not a luxury good.

Morphology of the Harderian gland of the Gecko, Tarentola mauritanica.

The Harderian gland of the gecko, Tarentola mauritanica, was studied at the histological, histochemical, and ultrastructural levels. It is a nonlobate compound acinar gland surrounded by a thin capsule of connective tissue. Numerous connective tissue-type mast cells, ultrastructurally similar to those described in other higher vertebrates, were identified in the interstitial tissue between the acini. Pyramidal or columnar-shaped secretory glandular cells were observed in the acini. In the glandular cells, two types of structures could be distinguished on the basis of their high or low electron density. Lipid droplets were found in the cytoplasm of the Harderian gland of both sexes. Histochemical tests showed that the Harderian gland of the gecko is a seromucous gland. The secretion is essentially merocrine, although an apocrine type of secretion is sometimes observed.

Organogenesis of the orbital glands in the lizard Podarcis s. sicula: a histological, histochemical and ultrastructural study.

The orbital glands of the lizard Podarcis s. sicula are represented by the anterior and posterior lacrimal glands and the Harderian gland. The anlage of the Harderian gland appears on about the 22nd day of development in the form of a short tubule projecting from the conjunctival epithelium. This event is coincident with the appearance of the nictitating membrane. At this stage the mesenchymal cells surrounding the glandular blastema proliferate at a high rate and form a definite sac, later occupied by both the Harderian gland and the anterior lacrimal glands. At the 26th day of development, the glandular blastema forms acini at its distal end. The prospective glandular cells are not yet differentiated histologically. At the 36th day of development, differentiated serous glandular cells become visible. At the 41st day of development, the acini fill up the preformed mesenchymal sac. Only at this stage does the most medial part of the gland differentiate into mucous-secreting anterior lacrimal gland. At the same time, a small primordium of the posterior lacrimal gland can be seen in the posterior commissure of the eye. The appearance of junctional complexes between epithelial cells and mesenchymal cells in the early developmental stages supports the role of the mesenchyme in the differentiation of the glandular cells. Since the glandular anlage differentiates laterally into Harderian gland and medially into anterior lacrimal gland, spatial and temporal differences seem to exist in the inductive process. Furthermore, a concentration gradient of the inductive substance(s) may be envisaged, since an intermediate zone is present between the Harderian gland and the anterior lacrimal gland, consisting of mixed glandular cells containing both mucous and serous secretory granules.

The effects of testosterone and estradiol on mast cell number in the harderian gland of the frog, Rana esculenta.

The Harderian gland (HG) of the frog Rana esculenta contains mast cells in the interstitial tissue. The mast cell number (MCN) is influenced by sex hormones. Gonadectomy in both sexes provoked a decrease in MCN in January, while no effect was observed in September. Sex hormone-replacement therapy gave different results; estradiol treatment in castrated males and females always increased MCN, while testosterone did not. Acute estradiol treatment provoked an increase in MCN on days 2 and 4 of treatment and the morphology of the glandular compartment appeared normal. On days 8, 10 and 12 of treatment the MCN drastically decreased. The majority of glandular acini appeared strongly disorganized and the interstitial tissue became hypertrophic in concomitance with an increased vascularization. Our results suggest that estradiol acts by stimulating mast cells and acute estradiol treatment provokes proliferation of interstitial connective tissue together with glandular cells damage.

The effect of sex hormones on lipid content and mast cell number in the harderian gland of the female toad, Bufo viridis.

The Harderian gland of the toad Bufo viridis is a dimorphic gland owing to the presence of lipid droplets in the female glandular cells present only during summer months. Ovariectomy causes the disappearance of sudanophilia and estrogen-treatment completely prevents this change, while testosterone-injection has little effect. Estradiol-treatment also provokes a proliferation of the interstitial connective tissue concomitantly with the mast cell number increase. Our results suggest that estradiol acts, stimulating both mast cell and connective tissue proliferation, and plays a role in determining the expression of the female type of the toad Harderian gland.

The determinants of the public-private mix in Canadian health care expenditures: 1975-1996.

The health care policy issue regarding the balance between public and private health spending is examined. An empirical model of the determinants of the public-private mix in Canadian health care expenditures over the period 1975-1996 is estimated for total health care expenditures as well as separate expenditure categories such as hospitals, physicians and drugs. The results find that the key determinants of the split are per capita income, government transfer variables and the share of individual income held by the top quintile of the income distribution. Much of the public-private split is determined by long term economic forces. However, the importance of the federal health transfer variables and the variables representing shifts in fiscal transfer regimes suggest the increase in the private share of health spending since 1975 is also partly the result of the policy choice to reduce federal health transfers.

TSH and thyroid hormones induce the release of secretory granules in the harderian gland of hypophysectomized frogs, (Rana esculenta): morphological observations.

The secretory activity of the harderian gland (hg) in the frog Rana esculenta is influenced by pituitary and thyroid hormones. Administration of thyroid stimulating hormone (TSH), T3 and T4 to hypophysectomized male and female frogs, affects the release of secretory granules in the lumina of glandular acini. In particular, treatment with T3 radically modified morphological parameters (height and area of acinar lumina of the glandular cells); T4 or TSH had less effect. Administration of thiouracil counteracted TSH effect, restoring the secretory content of the glandular cells. Ultrastructural observations confirmed these results. Our data suggest the existence of a thyroid-hg interaction and that thyroid hormones may be involved in the mechanism of release of mature secretory granules in the hg of the frog, Rana esculenta.

Mallory stain may indicate differential rates of RNA synthesis: II. Comparative observations in vertebrate nuclei.

The differential staining of nuclei by the use of the Mallory trichrome method was investigated in a variety of tissues of representative vertebrates. By this method nuclei stained orange or blue; erythrocyte nuclei stained red. Since the higher affinity for aniline blue is due to an increased RNA synthesis, it was possible to reveal not only the changing metabolic status of a cell type, as shown for instance in the liver parenchyma and other glandular tissues, and nervous tissue, but also in different cell populations in the same tissue, such as the spleen.