Long-term follow-up of boys who have undergone a testicular biopsy for fertility preservation.

STUDY QUESTION: What is the long-term reproductive health outcome of patients who have undergone testicular sampling for fertility preservation (FP) before and during the pubertal transition period? SUMMARY ANSWER: In long-term follow-up after testicular sampling for FP, hormonal data showed that 33% of patients had primary seminiferous tubule insufficiency (high FSH) while semen analyses showed 52% of patients having a severe reduction in total sperm counts or complete absence of ejaculated sperm. WHAT IS KNOWN ALREADY: During childhood and adolescence, both treatments for cancer and benign haematological diseases that require a bone marrow transplantation, can be detrimental to spermatogenesis by depleting the spermatogonial stem cell population. A testicular biopsy prior to chemotherapy or radiotherapy, even though still an experimental procedure, is now recommended for FP by European and USA oncofertility societies if performed within an institutional research setting. While short-term follow-up studies showed little to no post-operative complications and a normal testicular development after 1 year, data regarding the long-term follow-up of boys who have undergone this procedure are still lacking. STUDY DESIGN, SIZE, DURATION: This is a longitudinal retrospective cohort study that reports on the long-term follow-up of pre- and peri-pubertal boys who have undergone a testicular biopsy for FP between May 2005 and May 2020. All the patients included in this study were referred to our programme by haematologists-oncologists who are part of a regional multi-centric collaborative care pathway. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 151 boys referred to our FP programme, 139 parents/legal guardians accepted that their child undergo a testicular biopsy. Patient characteristics (i.e. age at biopsy, urogenital history, pubertal status at diagnosis), indications (disease type and dosage of gonadotoxic treatments), operative and post-operative data (biopsy volume, surgical complications), anatomopathological analyses (presence/absence of spermatogonia, Johnsen score) and reproductive data (semen analyses, FSH, LH, testosterone levels) were collected from the institutions' FP database and medical records or from the 'Brussels Health Network'. Cumulative alkylating agent treatment was quantified using the cyclophosphamide equivalent dose (CED). Patients who were 14 years or older at the time of the follow-up and in whom the testicular tissue was shown to contain spermatogonia were included in the reproductive outcome analysis. Comparison of the sperm count findings (absence/presence of spermatozoa) and FSH levels (high (≥10 IU/l)/normal) between patients who were either pre- (Tanner 1) or peri-pubertal (Tanner >1) at the time of the biopsy was done using the Mann-Whitney U or Fisher's tests. A multiple logistic regression was used to study the relationship between the hormone reproductive outcome (high versus normal FSH), as a proxy marker for fertility, and both the pubertal status (Tanner 1 versus Tanner >1) and Johnsen score at the time of the biopsy, while adjusting for CED. MAIN RESULTS AND THE ROLE OF CHANCE: A testicular biopsy was performed in 139 patients either before (129/139) or after (10/139) the start of a gonadotoxic treatment. Post-operative complications occurred in 2.1% (3/139). At the time of the procedure, 88% (122/139) of patients were pre-pubertal and 12% (17/139) were peri-pubertal. The presence of spermatogonia was documented in 92% (128/139) of cases. Follow-up data were available for 114 patients after excluding 23 deceased and two patients lost to follow-up. A paediatric endocrinologist's follow-up including clinical examination and data on reproductive hormones was available for 57 patients (age ≥14) and 19 (33%) of these were found to have high FSH levels (20 ± 8.8 IU/l). There were 37 patients who had returned to the reproductive specialist's consultation for post-treatment fertility counselling and results on semen analysis were available in 27 of these cases; 14/27 (52%) had severely impaired semen parameters including 8 who were azoospermic. Among patients who received an alkylating agent-based treatment (n = 42), a peri-pubertal status (Tanner >1) at the time of diagnosis/biopsy was found to be associated with a higher risk of having primary testicular failure (defined by an FSH ≥ 10 IU/l) after treatment completion with an OR of 6.4 (95% CI 1.22-33.9; P = 0.03). Of all the patients, 2.6% had already fulfilled their wish to build a family or were actively seeking parenthood. LIMITATIONS, REASONS FOR CAUTION: Although this is the largest cohort with follow-up data providing proxy markers of the reproductive potential of boys in whom a testicular biopsy for FP was performed before puberty or during the pubertal transition period, the amount of data provided is limited, and originating from a single programme. Further data collection to confirm the observations in other settings is therefore awaited. WIDER IMPLICATIONS OF THE FINDINGS: Testicular sampling for FP should be offered to boys at risk of losing their fertility (and is recommended for those at high risk) as part of ethically approved research programmes. Long-term follow-up data on increasing numbers of boys who have undergone an FP procedure will help improve patient care in the future as patient-specific factors (e.g. urogenital history, age at gonadotoxic therapy) appear to influence their reproductive potential after gonadotoxic therapies. STUDY FUNDING/COMPETING INTEREST(S): FNRS-Télévie, the Salus Sanguinis Foundation and the Belgian Foundation against Cancer supported the studies required to launch the FP programme. The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

In vitro formation of the blood-testis barrier during long-term organotypic culture of human prepubertal tissue: comparison with a large cohort of pre/peripubertal boys.

STUDY QUESTION: How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? SUMMARY ANSWER: The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. WHAT IS KNOWN ALREADY: Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. STUDY DESIGN, SIZE, DURATION: Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long-term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical files. A longitudinal analysis of connexin 43, claudin 11 and AMH expressions on immunohistological sections of organotypic cultured testicular tissue from three prepubertal boys who underwent a biopsy for fertility preservation was performed. Immunostaining was evaluated at culture Days 0, 1, 3, 10, 16, 27, 32, 53, 64 and 139 for two different types of culture media. MAIN RESULTS AND THE ROLE OF CHANCE: Immunohistochemical control sections showed progressive maturation of SCs, as shown by the decrease in AMH expression, with increasing age (P ≤ 0.01) and the AMH expression was negatively correlated with the expression of connexin 43 and claudin 11 (P ≤ 0.01 for both proteins). Androgen receptor (AR) expression increased with age (P ≤ 0.01) and was significantly correlated with the expression of connexin 43 (P = 0.002) and claudin 11 (P = 0.03). A statistical correlation was also found between the reduction of AMH expression and both the advancement of Tanner stages (P ≤ 0.01) and the differentiation of germ cells (P ≤ 0.01). Furthermore, positive correlations between BTB formation (using connexin 43 and claudin 11 expression) and age (P ≤ 0.01 for both the proteins), higher Tanner stages (P ≤ 0.001 and P ≤ 0.01 for connexin 43 and claudin 11, respectively), and presence of more advanced germ cells (P ≤ 0.001 for both proteins) were observed. In the subanalysis on organotypic cultured ITT, where a significant decrease in AMH expression as a marker of SC maturation was already reported, we showed the onset of expression of connexin 43 at Day 16 (P ≤ 0.001) and a constant expression of claudin 11 from Days 0 to 139, for all three patients, without differences between the two types of culture media. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Accessibility of prepubertal human testicular tissue is a major limiting factor to the analysis of cultured tissue samples from a wide number of patients, as would be needed to assess the in vitro development of the BTB according to the age. The impossibility of performing longitudinal studies on in vivo BTB formation in the same patient prevents a comparison of the time needed to achieve effective BTB formation and protein expression patterns in vivo and in vitro. WIDER IMPLICATIONS OF THE FINDINGS: To the best of our knowledge, this is the first report describing the expression of two BTB proteins in samples from a cohort of prepubertal and peripubertal boys, for the in vivo pattern, and in cultured ITT from a few prepubertal boys, for the in vitro evaluation. Since the formation of this barrier is essential for spermatogenesis and because little is known about its protein expression patterns and development in humans, a deeper understanding of the testicular microenvironment is essential to improve ITT in vitro culture conditions. The final aim is to restore fertility by acheiving in vitro differentiation of spermatogonial stem cells, using cryopreserved ITT collected before gonadotoxic therapies. STUDY FUNDING AND COMPETING INTEREST(S): Funding was received from Fonds National de la Recherche Scientifique de Belgique (Grant Télevie Nos. 7.4554.14F and 7.6511.16) and Fondation Salus Sanguinis. No conflict of interest has to be disclosed.

Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue.

STUDY QUESTION: Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)? SUMMARY ANSWER: Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation. WHAT IS KNOWN ALREADY: Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans. STUDY DESIGN, SIZE, DURATION: The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm3 tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either medium. GDNF was expressed until Day 139, demonstrating SC functionality. Moreover, a significant reduction in AMH expression and release indicated SC maturation. Testosterone concentrations in supernatants increased in both culture media, demonstrating LC functionality with paracrine interactions. SG were present up to Day 139, although the ratio between MAGE-A4-positive cells and well-preserved tubules was significantly reduced over the course of culture (P ≤ 0.001). SCs exhibited a decreased proliferation rate over time (P ≤ 0.05). The proliferation rate of SG remained stable until Day 64, but over the total culture period (139 days), it was found to have decreased (P ≤ 0.05). The number of apoptotic cells did not vary during culture, nor was any statistical difference observed between the two culture media for any of the studied parameters. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Loss of SG constitutes a limitation for evaluating full functionality of spermatogonial stem cells and warrants further investigation. The scarcity of human immature material is the reason for the limited amount of tissue available for experiments, precluding more comprehensive analysis. WIDER IMPLICATIONS OF THE FINDINGS: Our culture system, mimicking the peripubertal testicular microenvironment with SC maturation, LC functionality and preserved paracrine interactions, and the first to use human ITT, opens the door to a deeper understanding of niche and culture conditions to obtain sperm from cryostored ITT, with the ultimate goal of restoring fertility after gonadotoxic treatments. STUDY FUNDING/COMPETING INTERESTS: This project was supported by a grant from the Fond National de la Recherche Scientifique de Belgique (grant Télevie N° 7.4554.14F and N° 7.4512.15F) and the Fondation Salus Sanguinis. No conflict of interest is declared.

[Testicular tissue vitrification: evolution or revolution?]. / Vitrification du tissu testiculaire : évolution ou révolution ?

Preservation of reproductive health is a major concern for patient long-term quality of life. While sperm freezing has proven to be effective to preserve fertility after puberty, cryopreservation of immature testicular tissue (ITT) is emerging as a promising approach for fertility preservation in young boys. Slow-freezing (SF) is the conventional method used to preserve ITT and has resulted in the birth of mice offspring. In humans, methods to preserve ITT are still at the research stage. Controlled SF using dimethyl sulfoxide showed preservation of proliferative spermatogonia after thawing in a xenotransplantation model used to evaluate the efficiency of freezing and thawing procedures. However, spermatogonial recovery was low and normal differentiation could not be achieved. Both freezing/thawing and the environment of the xenotransplantation model may be implicated. Indeed, with SF, ice crystal formation could damage tissue and cells. For this reason, vitrification, leading to solidification of a liquid without crystallization, may be a promising alternative. ITT vitrification has been investigated in different species and shown spermatogonial survival and differentiation to the round or elongated spermatids stage. Offspring were also recently obtained after vitrification and allotransplantation in avians, confirming the potential of vitrification for fertility preservation. In humans, vitrification appears to be as efficient as SF in terms of spermatogonial survival and initiation of differentiation after xenotransplantation. However, before validation of such fertility preservation methods, completion of normal spermatogenesis and the fertilization capacity of sperm retrieved from cryopreserved and transplanted tissue should be fully investigated.

Autophagy and phagocytosis-like cell cannibalism exert opposing effects on cellular survival during metabolic stress.

Understanding mechanisms controlling neuronal cell death and survival under conditions of altered energy supply (e.g., during stroke) is fundamentally important for the development of therapeutic strategies. The function of autophagy herein is unclear, as both its beneficial and detrimental roles have been described. We previously demonstrated that loss of AMP-activated protein kinase (AMPK), an evolutionarily conserved enzyme that maintains cellular energy balance, leads to activity-dependent degeneration in neuronal tissue. Here, we show that energy depletion in Drosophila AMPK mutants results in increased autophagy that convincingly promotes, rather than rescues, neurodegeneration. The generated excessive autophagic response is accompanied by increased TOR and S6K activity in the absence of an AMPK-mediated negative regulatory feedback loop. Moreover, energy-depleted neurons use a phagocytic-like process as a means to cellular survival at the expense of surrounding cells. Consequently, phagocytosis stimulation by expression of the scavenger receptor Croquemort significantly delays neurodegeneration. This study thus reveals a potentially novel strategy for cellular survival during conditions of extreme energy depletion, resembling xeno-cannibalistic events seen in metastatic tumors. We provide new insights into the roles of autophagy and phagocytosis in the neuronal metabolic stress response and open new avenues into understanding of human disease and development of therapeutic strategies.

Psoas abscess complicating endovascular aortic aneurysm repair.

Aortic stent graft infection is a rare but serious complication associated with high mortality. This report emphasizes the need for continued awareness of potential graft-related septic complications in patients undergoing Endovascular Aortic Repair (EVAR). We report a case in which a post-EVAR patient became unwell about 30 days post operatively and was shown on CT scanning to have a psoas abscess. The abscess was managed with percutaneous drainage and antibiotics. The patient remains well with no evidence of psoas collection or perigraft infection one year on. We review the available literature and discuss the merits of different management strategies.

Vitrification of non-human primate immature testicular tissue allows maintenance of proliferating spermatogonial cells after xenografting to recipient mice.

This study demonstrates preservation of tissue integrity, maintenance of proliferating spermatogonia and Leydig cell functionality after vitrification and transplantation of non-human primate immature testicular tissue. The objective was to assess the potential of vitrification of non-human primate immature testicular tissue (ITT) in an in vivo xenotransplantation model. Testicular tissue was obtained from one immature rhesus monkey (Macaca mulatta) aged 4 years. Collection and vitrification of testicular tissue, followed by short-term xenografting (3 wks) to nude mice were performed to evaluate and compare vitrified/warmed and fresh tissue. Fresh ungrafted tissue was used for control purposes. Cell density and seminiferous tubule (ST) integrity were assessed by light microscopy. Presence of spermatogonia (SG) (MAGE-A4), proliferation (Ki-67) and Leydig cell (LC) functionality (3ß-hydroxysteroid dehydrogenase; 3ß-HSD) were evaluated by immunohistochemistry (IHC). Qualitative analysis revealed preservation of the histologic characteristics of SG and Sertoli cells (SCs), as well as cell-cell cohesion and cell adhesion to the basement membrane, in both vitrified and fresh grafted tissues. Survival of SG able to proliferate and functional LCs was confirmed by IHC in fresh and vitrified grafts. In conclusion, vitrification appears to be a promising approach, representing an alternative strategy to slow-freezing in the emerging field of ITT cryopreservation and cryobanking.

A description of CT features of Clostridium difficile infection of the small bowel in four patients and a review of literature.

Clostridium difficile associated disease is an increasingly common cause of morbidity and mortality. Pseudomembranous colitis following hospital-administered antibiotic treatment is the most common symptomatic manifestation. Small bowel enteritis caused by C. difficile, however, is rarely described. Here, we present a series of four patients with hospital-acquired small bowel enteritis caused by C. difficile, discuss its CT and histopathological features, and review the current literature.

Neuroendocrinological and molecular aspects of insect reproduction.

This review summarizes recent advances and novel concepts in the area of insect reproductive neuroendocrinology. The role of 'classic' hormones, such as ecdysteroids and juvenoids, to control reproduction is well documented in a large variety of insect species. In adult gonads, ecdysteroids appear to induce a cascade of transcription factors, many of which also occur during the larval molting response. Recent molecular and functional data have created opportunities to study an additional level of regulation, that of neuropeptides, growth factors and their respective receptors. As a result, many homologs of factors playing a role in vertebrate reproductive physiology have been discovered in insects. This review highlights several neuropeptides controlling the biosynthesis and release of the 'classic' insect hormones, as well as various peptides and biogenic amines that regulate behavioural aspects of the reproduction process. In addition, hormone metabolizing enzymes and second messenger pathways are discussed with respect to their role in reproductive tissues. Finally, we speculate on future prospects for insect neuroendocrinological research as a consequence of the recent 'Genomics Revolution'.

Glucocorticoid-inducible gene expression vectors for use in Drosophila melanogaster Schneider 2 cells.

Inducible, vector-based, expression systems that allow fine control of transgene expression are gaining more and more use in fundamental research as well as in therapeutic applications. In an effort to develop a tightly regulated heterologous expression system for Drosophila Schneider 2 cells, three different inducible reporter constructs were compared. These comprised six copies of the glucocorticoid response element fused to one of three distinct types of Drosophila gene promoters: (1) a TATA-box containing, (2) a TATA-less and (3) a bidirectional core sequence. These were fused to a luciferase reporter gene. The promoter constructs displayed different basal as well as agonist-induced activities. The implications of the observations made are discussed in the context of promoter properties and of induction of genes that may be studied in Drosophila.

An endothelin-converting enzyme homologue in the locust, Locusta migratoria: functional activity, molecular cloning and tissue distribution.

Endothelin-converting enzyme is the key enzyme in the process of endothelin production. Endothelin is a peptide that plays an important role in vasoconstriction and the development of neural crest-derived cells in vertebrates. Activity assays performed on membrane extracts from Locusta migratoria brain revealed the existence of a protease activity responsible for the formation of mature endothelin-1 from its precursor, big endothelin. Cloning experiments led to a cDNA sequence (Lom ECE) with an open reading frame of 727 amino acid residues displaying all the characteristic ECE features. A comparison of ECE activity levels among different tissues of the locust showed a high enzyme activity in the gonads and midgut. RT-PCR experiments showed a wide tissue distribution of Lom ECE mRNA, with transcription being most abundant in brain tissue.

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides.

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells.

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin. None of these amines (up to 0.1 mM) was able to reduce forskolin-stimulated cyclic AMP production in S2 cells. Furthermore, no agonist-induced calcium responses were observed. Nevertheless, the phenolamines octopamine (OA) and tyramine (TA) induced a dose-dependent increase of cyclic adenosine monophosphate (AMP) production in S2 cells, while serotonin, histamine, dopamine and melatonin (up to 0.1 mM) did not. The pharmacology of this response was similar to that of the octopamine-2 (OA2) receptor type. In addition, this paper provides evidence for the presence of an endogenous mRNA encoding an octopamine receptor type in these cells, which is identical or very similar to OAMB. This receptor was previously shown to be positively coupled to adenylyl cyclase.

Functional expression of a locust tyramine receptor in murine erythroleukaemia cells.

The LCR/MEL system (Locus Control Region/Murine Erythroleukaemia cells) was employed to express and characterize the Locusta migratoria tyramine receptor (TyrLoc), an insect G protein-coupled receptor. Functional agonist-dependent responses were recorded in stable, tyramine receptor expressing cell clones (MEL-TyrLoc). Tyramine elicited a dose-dependent increase of cytosolic Ca2+-ions and an attenuation of forskolin-induced cyclic adenosine monophosphate (AMP) production. Octopamine was shown to be a weak agonist for both responses. In addition, yohimbine proved to be a potent tyramine receptor antagonist. This study reports the first application of the LCR/MEL expression system in functional assays for G protein-coupled receptors and therefore expands the capabilities of this system by exploiting the functionality of the signal transduction pathways.

Tyramine injections reduce locust viability.

In the locust nervous system, tyramine is the direct precursor for octopamine synthesis and, as an octopamine analogue, it can activate octopamine receptors. Furthermore, the identification of specific tyramine receptors in Locusta migratoria and Drosophila melanogaster suggests that it is an important transmitter or modulator candidate. In this paper, we report that repeated tyramine injections reduced the viability of last instar larvae of Locusta and Schistocerca. In addition, a retardation of the last ecdysis was observed as a sublethal effect of the repeated tyramine treatment. Moreover, egg deposition by adult females was also retarded and/or drastically reduced. These effects show similarity to sublethal effects described for certain "insecticidal" octopamine receptor agonists, such as formamidines and phenyliminoimidazolidines. Since certain formamidine compounds were also shown to be agonists for the cloned tyramine receptors, it cannot be excluded that some lethal or sublethal consequences of tyramine administration are the result of an interaction with specific tyramine receptors.

Characterization of a receptor for insect tachykinin-like peptide agonists by functional expression in a stable Drosophila Schneider 2 cell line.

STKR is an insect G protein-coupled receptor, cloned from the stable fly Stomoxys calcitrans. It displays sequence similarity to vertebrate tachykinin [or neurokinin (NK)] receptors. Functional expression of the cloned STKR cDNA was obtained in cultured Drosophila melanogaster Schneider 2 (S2) cells. Insect tachykinin-like peptides or "insectatachykinins," such as Locusta tachykinin (Lom-TK) III, produced dose-dependent calcium responses in stably transfected S2-STKR cells. Vertebrate tachykinins (or neurokinins) did not evoke any effect at concentrations up to 10(-5) M, but an antagonist of mammalian neurokinin receptors, spantide II, inhibited the Lom-TK III-induced calcium response. Further analysis showed that the agonist-induced intracellular release of calcium ions was not affected by pretreatment of the cells with pertussis toxin. The calcium rise was blocked by the phospholipase C inhibitor U73122. In addition, Lom-TK III was shown to have a stimulatory effect on the accumulation of both inositol 1,4,5-trisphosphate and cyclic AMP. These are the same second messengers that are induced in mammalian neurokinin-dependent signaling processes.

Tachykinin-like peptides and their receptors. A review.

Tachykinin-like peptides have been identified in many vertebrate and invertebrate species. On the basis of the data reviewed in this paper, these peptides can be classified into two distinct subfamilies, which are recognized by their respective sequence characteristics. All known vertebrate tachykinins and a few invertebrate ones share a common C-terminal sequence motif, -FXGLMa. The insect tachykinins, which have a common -GFX1GX2Ra C-terminus, display about 30% of sequence homology with the first group. Tachykinins are multifunctional brain/gut peptides. In mammals and insects, various isoforms play an important neuromodulatory role in the central nervous system. They are involved in the processing of sensory information and in the control of motor activities. In addition, members of both subfamilies elicit stimulatory responses on a variety of visceral muscles. The receptors for mammalian and insect tachykinins show a high degree of sequence conservation and their functional characteristics are very similar. In both mammals and insects, angiotensin-converting enzyme (ACE) plays a prominent role in tachykinin peptide metabolism.

Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants.

As a first step toward the exploitation of the disaccharide trehalose as a stress-protective and preservative agent in plants, we engineered trehalose biosynthesis in tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) by introducing the otsA and otsB genes from Escherichia coli, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. In leaves of transgenic tobacco plants, very low levels of trehalose accumulation were obtained (0.11 mg g-1 fresh weight), whereas in transgenic potato tubers, no trehalose accumulated at all. Plant trehalase activity was shown to affect the accumulation of trehalose in these plants. An increase in trehalose accumulation, up to 0.41 and 4.04 mg g-1 fresh weight in tobacco leaves and potato micro-tubers, respectively, was noted when the potent trehalase inhibitor validamycin A was added to in vitro plants and to hydroponically grown greenhouse plants. Stunted growth and the formation of lancet-shaped leaves by trehalose-accumulating tobacco plants suggest a negative effect of trehalose biosynthesis on N. tabacum development. It is surprising that experiments with wild-type plants cultured in the presence of validamycin A indicate that, despite current belief, the capacity to synthesize trehalose may not be restricted to primitive phyla of vascular plants and certain "resurrection plants," but may exist throughout the angiosperms.