[Cerebral metabolic changes and chronic back pain: Study taking into consideration clinical and psychological parameters]. / Hirnmetabolische Veränderungen bei chronischem Rückenschmerz : Studie unter Berücksichtigung von klinischen und psychischen Parametern.

BACKGROUND: The manifestation of chronic pain and psychological impairments are related to alterations of neurotransmitter metabolism in cerebral pain processing regions, e.g., anterior cingular cortex (ACC), insula. Magnetic resonance spectroscopy ((1)H-MRS) enables in vivo quantification of neurotransmitters in the brain and was applied in this study to examine the hypothesized chronic pain-related imbalance between excitatory (glutamatergic) and inhibitory (GABA-ergic) neurotransmitter turnovers in the brain of patients with nonspecific chronic pain. MATERIALS AND METHODS: A total of 19 patients with nonspecific chronic (> 3 months) back pain and 19 age- and gender-matched healthy subjects participated in this study. Glutamate and GABA as well as glutamate/GABA ratios were determined in the ACC and insula using (1)H-MRS. Sociodemographic, psychological, and pain-related features were measured with standardized questionnaires. RESULTS: There was a strong variance of glutamate/GABA ratios for both patients and healthy subjects with no significant difference between the two groups. Regression analysis revealed certain significant predictors, such as anxiety as causal variable for reduced glutamate and depression and age as predictors for reduced GABA in ACC. In the patient group, intensity of pain was a significant predictor for glutamate and GABA levels in the insula. CONCLUSIONS: Despite the uniform diagnosis of nonspecific chronic back pain, we observed a strong variance of neurotransmitters in cerebral pain processing regions. It is necessary to include psychological as well as clinical parameters (e.g., intensity of pain or depression) for a proper interpretation of neurotransmitter turnovers.

Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress.

Oxidative stress in the male germ line is known to be a key factor in both the etiology of male infertility and the high levels of DNA damage encountered in human spermatozoa. Because the latter has been associated with a variety of adverse clinical outcomes, including miscarriage and developmental abnormalities in the offspring, the mechanisms that spermatozoa use to defend themselves against oxidative stress are of great interest. In this context, the male germ line expresses three unique forms of thioredoxin, known as thioredoxin domain-containing proteins (Txndc2, Txndc3, and Txndc8). Two of these proteins, Txndc2 and Txndc3, retain association with the spermatozoa after spermiation and potentially play an important role in regulating the redox status of the mature gamete. To address this area, we have functionally deleted the sperm-specific thioredoxins from the male germ line of mice by either exon deletion (Txndc2) or mutation of the bioactive cysteines (Txndc3). The combined inactivation of these Txndc isoforms did not have an overall impact on spermatogenesis, epididymal sperm maturation, or fertility. However, Txndc deficiency in spermatozoa did lead to age-dependent changes in these cells as reflected by accelerated motility loss, high rates of DNA damage, increases in reactive oxygen species generation, enhanced formation of lipid aldehyde-protein adducts, and impaired protamination of the sperm chromatin. These results suggest that although there is considerable redundancy in the systems employed by spermatozoa to defend themselves against oxidative stress, the sperm-specific thioredoxins, Txndc2 and Txndc3, are critically important in protecting these cells against the increases in oxidative stress associated with paternal age.

Intraperitoneal inflammation decreases endometriosis in a mouse model.

BACKGROUND: The role of the immune system in the pathogenesis of endometriosis remains elusive. It has been shown that patients have an altered peritoneal environment with increased levels of inflammatory cytokines, activated macrophages and reduced clearance of retrogradely transported endometrial fragments. However, it is not known if this unique inflammatory situation is cause or consequence of endometriosis. This study investigates the impact of a pre-existing peritoneal inflammation on endometriosis establishment in a mouse model. METHODS: Endometriosis was induced by intraperitoneal injection of enhanced green fluorescent protein (EGFP)-expressing endometrium in mice. In parallel, a peritonitis model was established via intraperitoneal injection of thioglycolate medium (TM). Finally, endometriosis was induced in the inflamed peritoneal cavity and lesion establishment as well as morphological and histological characteristics were analysed. RESULTS: Induction of endometriosis in an inflamed peritoneal cavity resulted in fewer lesions and significantly lower sum of lesion surface area per mouse in the TM-treated group. Additionally, a higher amount of non-attached debris could be detected in the peritoneal cavity of TM-treated mice. CONCLUSIONS: An intraperitoneal inflammation decreases endometriosis establishment in this mouse model. Thus, a pre-existing peritoneal inflammation might not be a factor favouring the development of endometriosis.

The AMPPA network as a successful model for public-private or private-private partnership.

In 1997, the Rockefeller Foundation and the Ernst Schering Research Foundation (a subsidiary of Schering AG, Germany, on a non-profit basis) mounted a multi-year global collaborative effort, involving a network of top-level research institutions to intensify research on the regulation of the male reproductive system with special emphasis on post-testicular activity, utilizing new approaches in molecular pharmacology (application of molecular pharmacology for post-testicular activity (AMPPA) network). The new venture proved a success as a public-private sector partnership, as a collaborative scientific program, and as an approach to identify new targets applicable and suitable for drug finding for male fertility regulation.

Steroidal sigma receptor ligands affect signaling pathways in human spermatozoa.

In human spermatozoa, Ca(2+) entry is stimulated by progesterone or prostaglandin E(1) (PGE(1)). The regulation of cation currents by progestins involves sigma receptors, and sigma binding sites are abundant in testis. We examined the effects of sigma ligands on human spermatozoa. Ca(2+) entry induced by progesterone or PGE(1) was not altered by the sigma ligands haloperidol and ditolylguanidine. However, the steroidal sigma ligands RU 3117 and RU 1968 had distinct effects. Stimulation by RU 3117 resulted in activation and homologous desensitization of the sperm progesterone receptor but not of the PGE(1) receptor. Because haloperidol and ditolylguanidine did not affect RU 3117 and progesterone actions in spermatozoa, we conclude that sigma receptors are not involved. However, RU 1968 potently inhibited both the progesterone- and PGE(1)-induced Ca(2+) entry and acrosome reaction. At higher concentrations, RU 1968 also inhibited hormonal Ca(2+) signaling in fibroblasts. Despite suppression of Ca(2+) mobilization, inhibition of phospholipase C by RU 1968 was not observed. Furthermore, RU 1968 did not impair the binding of inositol-1,4,5-trisphosphate to its endoplasmic reticulum receptor. Because RU 1968 preferentially inhibits signaling pathways in spermatozoa, the future development of more selective drugs structurally related to RU 1968 may be a novel approach for pharmacological contraception.

Collaboration between industry and academia--prospects for male fertility control.

Drug development within the pharmaceutical industry is probably the field with the highest level of regulations. Due to the complexity of the different components of drug development and drug surveillance the need for a sophisticated organization and infrastructure is obvious. In addition, there is a necessity for sufficient resources and long-term commitment as well as logistic and long-term knowledge management. In order to secure high professional standards at all levels of this highly complex value creating chain, the number of cooperative arrangements in the pharmaceutical industry are increasing. The identification of new targets in the drug finding process calls in particular for outside partners. At the same time the preparedness of non-industrial researchers to cooperate with industry has also increased significantly. The area of fertility control, especially male fertility control, provides an excellent example for this kind of cooperation between industrial and non-industrial partners. Here a cooperative network is described which probably meets practically all relevant criteria for both the non-industrial but also the industrial partner. Some principles for the management of such a cooperative network are discussed. We believe that this kind of network can serve as a model for similar networks in other fields.

Effect of angiotensin converting enzyme (ACE) and angiotensins on human sperm functions.

The presence of components of the renin angiotensin system (RAS) and specific receptors of angiotensin II in the female and male reproductive tract supports the hypothesis that reproductive functions may be controlled by RAS. Therefore, the present study investigated the influence of ACE and angiotensins on sperm functions and the sperm-egg interaction. The experiments did not indicate direct effects of ACE on the capacitation process or acrosome reaction. Release of ACE from human spermatozoa during capacitation was not related to their ability to undergo acrosome reaction after stimulation with ionophore. Therefore, ACE release does not seem to be a useful clinical marker for human sperm capacitation. However, decreased binding of human spermatozoa to the oolemma of zonafree hamster oocytes after inhibition of ACE by captopril indicates that kininase II is involved in sperm-egg interactions. In contrast to other studies, incubation with captopril had no influence on sperm binding to the zona pellucida. Because effects of ACE on sperm-egg interactions but not on capacitation or acrosome reaction were observed, several experiments were performed to study the influence of substrates and products on the acrosome reaction. Angiotensin II induced the acrosome reaction dose-dependently, whereas angiotensin I had no effect on the acrosome reaction. The effect of angiotensin II on acrosome reaction seems to be calcium-dependent and mediated by protein kinases. Since a specific type 2 angiotensin II receptor inhibits the acrosome reaction induced by angiotensin II, this subtype of receptors may be present at the surface of sperm heads. Another clue for the presence of type 2 receptors on human spermatozoa is the finding that pertussis toxin did not inhibit the angiotensin II induced acrosome reaction. In contrast to type 1 angiotensin II receptors, type 2 receptors are known to be G-protein independent.

Zona pellucida as physiological trigger for the induction of acrosome reaction.

To evaluate the kinetics of acrosome reaction, sperm samples from four fertile donors were prepared by swim-up and incubated with solutions of human zonae containing 0.1, 0.15, 0.3, 0.5 and 1.0 zonae microliter-1. After 20, 40 and 60 min of incubation at 37 degrees C, aliquots were taken for evaluation of the acrosomal status. The results showed a distinct time- and dose dependence of the acrosome reaction induced by solubilized zona proteins. After 60 min of incubation in 1.0 zonae microliter-1, about 80% of the spermatozoa showed signs of acrosomal loss; about 40% were completely acrosome-reacted. In addition, zona-bound sperm showed the same ratios of acrosome-reacted spermatozoa in control experiments. The velocity of acrosome reaction was calculated by means of a double-reciprocal plot being 2.0-2.5% min-1 for completely reacted spermatozoa and those showing signs of acrosome reaction. However, both subgroups differed considerably in their constants of equilibrium (K = 2.0 ZP microliter-1 and K = 0.2 ZP microliter-1, respectively). In nonreacted and partly reacted spermatozoa results might indicate a disturbed course of acrosome reaction or possibly the existence of different subpopulations in respect of sperm competition.

Characterization of mouse epididymal acrosin: comparative studies with acrosin from boar and human ejaculated spermatozoa.

Acrosin is an acrosomal protease believed to play a major role in fertilization. It is synthesized as an inactive precursor, proacrosin, which is processed via (auto)proteolysis into active form(s). In this paper, comparative studies on the characteristics of acrosin from mouse, boar and human are reported. The mouse proacrosin/acrosin was especially investigated to clarify whether or not the enzyme undergoes modifications during epididymal maturation. Acrosomal extracts from mature and immature mouse spermatozoa, as well as from ejaculated boar and human spermatozoa, were analysed by means of SDS-electrophoresis, Western blot and activity measurements. The studies showed that epididymal maturation produced a shift in the molecular weight of proacrosin. It was also observed that the activation kinetics differ strongly between the three species studied. Human proacrosin showed a constant substrate turnover, acrosin from boar showed sigmoidal activation kinetics and mouse acrosin, either from the caput or the cauda epididymides, showed a rapid decay in activity, suggesting the presence of an endogenous specific inhibitor.

Differential androgen regulation of the murine genes for cysteine-rich secretory proteins (CRISP).

The androgen dependency of the genes coding for the cysteine-rich secretory proteins (CRISP) was analysed in their main sites of expression. Male mice were treated with the gonadotropin-releasing hormone antagonist Ac-DNapAla-DClPhAla-DPyrAla-Ser-Tyr-DCtl-Leu-Lys (Mor)-Pro-DAla-NH2 [DNapAla, D-2-naphthyl-Ala; DClPhAla, D-4-chlorphenyl-Ala; DPyrAla, D-pyridyn-3-yl-Ala; DCtl, D-citrulline; Lys(Mor), L-2-amino-6-(morpholin-4-yl)-hexanoic acid], and CRISP RNA levels were assessed by northern blot and competitive reverse transcriptase-mediated (RT)-PCR. In the salivary gland, CRISP-1 and to a lesser extent CRISP-3 expression was markedly reduced, in spite of an up-regulation of androgen receptor transcript levels. A down-regulation of CRISP-1 expression was also observed in the epididymis. Conversely, the levels of the testicular CRISP-2 transcripts were hardly affected at all. Female mice were ovariectomised and treated with testosterone propionate, and their salivary gland RNAs analysed. CRISP-1 and CRISP-3 RNA levels were significantly increased, and these effects were prevented by a concomitant treatment with the antiandrogen flutamide. Androgen receptor transcript levels were not affected by androgen administration but increased following antiandrogen treatment. CRISP expression during postnatal development was monitored by northern blot analysis. CRISP-1 and CRISP-2 transcripts were detected as early as 22 days after birth in the epididymis and testis, respectively, whereas CRISP-3 mRNA was visible only from day 30 in the salivary gland. A sharp increase of all CRISP levels was noted on day 40, coincident with the onset of sexual maturity. Altogether these results indicate that despite their high similarity, the CRISP genes are differentially regulated by androgens.

Zona pellucida mediated acrosome reaction and sperm morphology.

Sperm samples from 29 men randomly selected from the andrology laboratory, were used to evaluate acrosome reaction response to solubilized human zona pellucida. Capacitated sperm samples were exposed to a solution containing 2 zona pellucidae (ZP) per microl for 60 min, after which acrosomal status were recorded using a PSA-FITC technique. Controls included samples supplied by fertile sperm donors. After completion of acrosome reaction studies, patient samples were divided according to the percentage of morphologically normal spermatozoa. Three basic groups were identified, namely, fertile donors, teratozoospermic (normal sperm morphology 5-14%; n = 25) and severely teratozoospermic (normal sperm morphology < 4%; n = 4) groups. The mean percent normal sperm were 15.8 +/- 0.9, 10.4 +/- 0.7 and 2.7 +/- 0.7, respectively, for normozoospermic donors, teratozoospermic and severely teratozoospermic men. The mean percentage (+/-SE) ZP mediated acrosome reacted sperm among teratozoospermic and severely teratozoospermic cases was 25.8% +/- 0.9 and 19.0% +/- 0.9 (P = 0.001), compared to 36.8% +/- 0.9 for the donor controls. Results were analysed and expressed as correlations between sperm morphology and acrosomal response to human solubilized zona pellucida, spontaneous and calcium ionophore induced acrosome reaction. Predictive values for acrosome responsiveness were depicted with ROC curve analyses. Sperm morphology evaluated by strict criteria correlated positively and highly significantly with the responsiveness of the acrosome reaction (r = 0.91, P = 0.0001). At a morphology cut-off value of 4%, the ROC curve analysis showed sperm morphology to be highly predictive of zona pellucida induced acrosome responsiveness with a sensitivity of 100% and negative predictive value of 100%. Spontaneous and calcium ionophore induced acrosome reactions revealed no correlation with sperm morphology. It was concluded that (i) morphological features of human spermatozoa are indicative of specific functional characteristics; (ii) zona pellucida induction of the acrosome reaction is superior, as a predictor of sperm morphology, compared to calcium ionophore induced and spontaneous acrosome reactions.

Inhibition of G protein in human sperm and its influence on acrosome reaction and zona pellucida binding.

OBJECTIVE: To evaluate human sperm acrosomal status, zona pellucida (ZP)-binding capacity, and sperm motion characteristics after treatment with pertussis toxin followed by exposure to increasing concentrations of solubilized human ZP. DESIGN: Prospective analytical study. SETTING: Normal human sperm donors in an academic research environment. INTERVENTION: Sperm were prepared with a wash and swim-up method and treated with a final concentration of 100 ng/mL pertussis toxin. Acrosomal status were determined using a Pisum sativum agglutinin-fluorescien-isothiocyanate method after exposure of sperm to 0.25, 0.5, 0.75, and 1.00 ZP/microL solutions of human ZP. Zona binding potential was recorded using intact zona-binding assays. Motion characteristics were recorded with a semen analyzer. MAIN OUTCOME MEASURE: Percentage acrosome-reacted sperm, number of zona-bound sperm, and sperm motion parameters. RESULTS: Spermatozoa treated with 100 ng/mL pertussis toxin, followed by ZP-mediated acrosome reaction induction, showed a significant decrease in the percentage of acrosome-reacted sperm compared with untreated controls. Motion characteristics of 3-hour capacitated sperm after treatment with either phosphate-buffered saline (PBS) or pertussis toxin were not different. Pertussis toxin-treated sperm populations bound significantly more sperm to the ZP after 4 hours incubation compared with the PBS-control groups: 137.1 +/- 8.0 compared with 96.3 +/- 7.0 (mean +/- SEM). CONCLUSIONS: The data support the concept of the controlling mechanism and importance of G proteins during the ZP-mediated acrosome reaction. Intact acrosome correlate with and are needed to ensure tight zona binding.

Development of new immunocontraceptives-industrial perspective.

Contraception has been practiced for thousands of years. Nevertheless, it took until the late 1950s and early 1960s before a major breakthrough in contraceptive technology was achieved by the introduction of oral hormonal contraceptives. However, we have not succeeded in the development of non-hormonal contraceptive that is comparable to the pill regarding its efficacy, safety, and acceptance. The immunological interference with the complex fertilization process is a very attractive target in this respect, whereby the zona pellucida, a non-cellular surrounding of all mammalian eggs, represents a potentially ideal target. Another interesting target are sperm: either for the development of a female contraceptive or for a male contraceptive, although the latter approach does not look very promising so far. In conclusion, given the enormous impact on mankind of a growing world population and given the very individual needs for contraceptive methods of different women in one and the same country and in different cultures we should make widely available a whole set of suitable, adjusted methods of fertility control and this includes the search for an effective method of male fertility control.

Defining bioassay conditions to evaluate sperm/zona interaction: inhibition of zona binding mediated by solubilized human zona pellucida.

OBJECTIVE: Our goal was to study the influence of solubilized human zonae pellucidae on zona binding potential and acrosome reaction. MATERIALS AND METHODS: Zona pellucida (ZP) solutions were prepared by dissolving zona in acidic buffer, NaH2PO4 (pH 2.5), to obtain 0.1 and 0.5 zona pellucida/microliters. Zona binding capacity was evaluated by the addition of oocytes (10-fold) to sperm/zona pellucida solution droplets. The number of sperm bound to each oocyte was recorded. Zona pellucida-mediated acrosome activity was evaluated after 60 min of coincubation of sperm and 0.5 ZP/microliters. RESULTS: The mean (+/- SE) number of sperm bound for control, 0.1 ZP/microliter, and 0.5 ZP/microliter was 181.2 +/- 12, 79.6 +/- 5, and 38.8 +/- 3, respectively. Zona pellucida-exposed sperm populations showed significant more acrosome-reacted sperm compared to control sperm, namely, 78 versus 32%, respectively (P = 0.001). CONCLUSIONS: The observed zona binding inhibition might be ascribed to zona receptor blocking on the sperm surface or the inability of acrosome-reacted sperm to bind to the zona pellucida.

Mouse androgen-dependent epididymal glycoprotein CRISP-1 (DE/AEG): isolation, biochemical characterization, and expression in recombinant form.

In the rat, the secretory glycoprotein DE/AEG is one of the main constituents of the epididymal fluid. We have recently reported the cloning of the cDNA for the related cysteine-rich secretory protein-1 (CRISP-1) from murine epididymis (Haendler et al., 1993; Endocrinology 133:192-198). The protein has now been isolated from the same organ and its N-terminal amino acid sequence has been determined. CRISP-1 exhibited an isoelectric point of approximately 6.8. High levels of CRISP-1 antigen were detected in the corpus and cauda of the epididymis, vas deferens, seminal vesicle, prostate, and in the salivary gland by immunohistochemistry. A quantitative analysis of the cauda epididymal fluid by sandwich ELISA revealed that CRISP-1 represented approximately 15% of the total protein. For heterologous expression, the CRISP-1 coding sequence was introduced into the pMPSV/CMV vector before transfection of baby hamster kidney (BHK) cells and selection with puromycin and neomycin. Expression in insect cells was achieved by co-transfection of Sf9 cells with a transfer vector and baculovirus DNA. Recombinant CRISP-1 was isolated in quantities sufficient for structural analysis. Ethyl maleimide treatment showed that all 16 cysteines were engaged in disulfide bonds. Proteolytic digestion demonstrated that the six cysteines localized in the N-terminal moiety formed three bonds with each other, suggesting the existence of two discrete domains in the protein.

Influence of elevated pH levels on structural and functional characteristics of the human zona pellucida: functional morphological aspects.

PURPOSE: A total of 86 fresh and salt-stored immature human oocytes derived from postmortem ovarian tissue were used for this study. METHODS: Oocytes were randomly incubated either in synthetic human tubal fluid medium (untreated zonae) or in a chemically defined medium (treated zonae). RESULTS: Sperm binding experiments using hemizona assay conditions exhibited a 10-fold increased binding of sperm to treated compared to untreated oocytes (272.7 +/- 43 versus 24.3 +/- 15 sperm bound, respectively; P < 0.0001). pH recordings during incubation showed elevated pH levels of 8.1 compared to pH 7.2 among treated and untreated zonae, respectively. Ultrastructural examination showed a spongy appearance of the surface of treated zonae, whereas untreated zonae appeared compact with smooth surface. CONCLUSIONS: The marked increase in sperm binding among treated zonae, together with the ultrastructural findings, suggest that the altered zona surface enhances sperm binding. The physiological maturational process of the zona pellucida might be manipulated in vitro, thus increasing sperm binding to the zona.

Management of benign prostatic hyperplasia with particular emphasis on aromatase inhibitors.

The pathogenesis of human benign prostatic hyperplasia (BPH) has not been fully elucidated. There is, however, evidence that estrogens--besides other factors--might play an important role for the growth of the prostate. Consequently, estrogen deprivation might be a new, useful principle for a conservative treatment of BPH. Atamestane, a new, highly selective steroidal aromatase inhibitor has been proven to be successful in antagonizing experimentally-induced estrogen-related stromal overgrowth of the prostate in dogs and monkeys. Double-blind placebo controlled studies are now underway in Europe and the U.S.A. It is anticipated that these studies will give us a definite answer of the clinical validity of this concept in BPH patients in the near future. However, it is very important to take into consideration that for an effective treatment of BPH, a reduction of both the glandular and stromal elements has to be achieved. In other words, both androgens and estrogens seem to be involved in the regulation of (over)growth of the prostate. Therefore, a combination of an androgen and estrogen deprivation might be a more promising approach than any single treatment.

Effects of estrogen deprivation on human benign prostatic hyperplasia.

Sex steroids are thought to play an essential role in the pathogenesis of human benign prostatic hyperplasia (BPH). Since recent studies in animal models and in men have shown that estrogens might be causally linked to the onset and maintenance of BPH, we examined the effect of 1-methyl-androsta-1,4-diene-3,17-dione (Atamestane), a newly developed aromatase inhibitor, in men with BPH. In an open multicenter study 49 men (mean age 70.1 years, range 55 to 84) with obstructive BPH were treated with atamestane (3 x 200 mg/day) for 3 months. Of the 49 patients 44 completed the treatment period; the other patients discontinued the study for reasons unrelated to treatment. With treatment BPH-related symptoms such as daytime voiding frequency, nycturia, peak flow and residual urine improved considerably; however, these parameters did not reach statistical significance. The mean prostatic volume decreased significantly from 74.2 +/- 31.7 to 64.0 +/- 31 ml (mean +/- SD). Serum estrogen levels decreased markedly during treatment. In addition intraprostatic estrogen concentration decreased with treatment as compared to estrogen levels in hyperplastic prostates from untreated patients. The following conclusions can be drawn from this study: first, estrogens appear to have an important supportive role in established BPH, and second, estrogen deprivation improved BPH-related symptoms and reduced significantly prostatic volume.