Yvetta Koeva

The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells

Previous studies have demonstrated local functions for neurotrophins in the developing and mature testis of rodents. To examine whether these signaling molecules are present and also potentially active in the human testis, we characterized immunohistochemically the expression and cellular localization of the known neurotrophins and their receptors during prenatal testicular development as well as in the adult human testis. Results obtained revealed the presence of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and 4, as well as neurotrophin receptors p75NTR, TrkA, TrkB, and TrkC during testis morphogenesis. These proteins were also detectable in the adult human testis, and their local expression could be confirmed largely by immunoblot and RT-PCR analyses. Remarkably, the Leydig cells were found to represent the predominant neurotrophin/receptor expression sites within both fetal and adult human testes. Functional assays performed with a mouse tumor Leydig cell line revealed that NGF exposure increases cellular steroid production, indicating a role in differentiation processes. These findings support previously-recognized neuronal characteristics of Leydig cells, provide additional evidence for potential roles of neurotrophins during testis morphogenesis and in the mature testis, and demonstrate for the first time a neurotrophin-induced functional activity in Leydig cells.

Catecholamine-synthesizing enzymes in the adult and prenatal human testis

Catecholamines play functional roles in the mature and developing mammalian testis but the cell types responsible for their local synthesis are still controversially discussed. Here, we demonstrate that four enzymes involved in the biosynthesis of catecholamines, namely, tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH) and phenylethanolamine- N-methyltransferase (PNMT), are expressed in Leydig cells of the human testis. Tyrosine hydroxylase, the key enzyme of the biosynthesis of catecholamines, was localized to Leydig cells both at the transcript level (by RT-PCR analyses and by in situ hybridization assays) and at the protein level (by immunoblotting and by immunohistochemistry). The other enzymes were also demonstrated in Leydig cells by RT-PCR and immunohistochemical analyses. The presence of TH, AADC, DBH, and PNMT in human Leydig cells was found, in addition, by immunohistochemical approaches carried out on sections from prenatal human testes. Thus, the present study identifies the Leydig cells as the presumed sites of catecholamine production in both the mature and fetal human testes and further supports the previously recognized neuroendocrine characteristics of this cell type.

Age-related changes in the expression of 11beta-hydroxysteroid dehydrogenase type 2 in rat Leydig cells.

Previous studies in rats have shown that the ability of Leydig cells (LCs) to produce testosterone significantly declines with age. To address the possible mechanisms by which aging LCs lose their steroidogenic function, we determined the effect of aging on the expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2. The enzyme plays a protective role in blunting the suppressive effects of glucocorticoids on LCs steroidogenesis. Our immunohistochemical analysis revealed progressive decline in 11beta-HDS type 2 expression in LCs of the 18 months of age rats and the most significant reduction in 11beta-HSD2 immunoreactivity was evident in the testicular interstitium of 24- month-old rats. The decrease in the 11beta-HDS type 2 immunostaining in LCs during aging coincided with decline in insulin-like 3/relaxin-like factor (INSL3/RLF) expression, an independent marker for LCs differentiation status. Concomitant with the age-related decrease of 11beta-HDS type 2 immunoreactivity in the LCs population, the immunoexpression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), marker for LCs steroidogenic activity, was greatly reduced at 24 months compared to 3-month-old control. Similar pattern of expression exhibited also androgen receptor (AR) which is localized in the nuclei of Sertoli cells (SCs), LCs, and peritubular cells. During ages we observed progressive decrease in the immunoreactivity for AR in the testicular types and there was a loss of stage specificity in SCs at age of 24 months. It now seems evident that a variety of factors are likely to be involved in age-related decreases in LCs steroidogenesis, including 11beta-HSD type 2. The observed reduction in 11beta-HSD type 2 expression in aging LCs reflects the decline in their protection ability, opposing the suppressive effect of glucocorticoids on testosterone production.

Brain-derived neurotrophic factor and its serum levels in schizophrenic patients.

ABSTRACT INTRODUCTION: Neurotrophins have an important role in regulating the development and maintenance of the peripheral and central nervous systems’ function. Thus, the neurotrophin hypothesis of schizophrenia has postulated that the changes in the brain of schizophrenic patients are the result of disturbances of developing processes involving these molecules. AIM: We analyse in the present study the changes in the serum levels of brain-derived neurotrophic factor (BDNF) in schizophrenic patients as possible epiphenomena of underlying alterations of the neurotrophic factor in central nervous system, reflecting its role in the pathophysiology of schizophrenia. PATIENTS AND METHODS: Twenty-one schizophrenic patients satisfying the DSM-IV criteria for diagnosis of schizophrenia were enrolled in the study. The control group consisted of 28 age-matched mentally healthy subjects. Serum BDNF levels were determined in patients and normal controls using ELISA (Chemicon International, USA & Canada). The data were analyzed statistically with Student’s t- test in SPSS 9.0. RESULTS: The serum BDNF levels were lower in the schizophrenic patients than in the control subjects, reaching statistically significant difference (t = 2.72, p = 0.009). Female patients had lower serum BDNF levels than the male patients but the difference fell short of statistical significance (t = 0.1, p = 0.9). CONCLUSIONS: The BDNF reduction in serum indicates a potential deficit in neurotrophic factor release in patients with schizophrenia and support the concept that BDNF might be associated with schizophrenia РЕЗЮМЕ ВВЕДЕНИЕ: Нейротрофины играют важную роль в процессе развития и в поддержке функций периферической и центральной нервной систем. Согласно нейротрофической гипотезе шизофрении изменения в мозгу пациентов с шизофренией являются результатом нарушений в процессе развития, в которые включены эти молекулы. ЦЕЛЬ: В настоящем исследовании авторы анализируют изменения в сывороточных уровнях BDNF у пациентов с шизофренией как вероятный эпифеномен предыдущих нарушений этого нейротрофического фактора в ЦНС, отражающие его роль в патофизиологии заболевания. ПАЦИЕНТЫ И МЕТОДЫ: 21 пациента (покрыты DSM-IV критерии диагноза „шизофрения”) включено в настоящее исследование. Контрольная группа состоит из 28 психически здоровых индивидов. Пациенты и здоровые индивиды одного и того же возраста. Сывороточные уровни BDNF определены иммунометрически с помощью ELISA анализа. Полученные данные анализированы Student’s t- test-ом в SPSS 9.0. РЕЗУЛЬТАТЫ: Сывороточные уровни BDNF у больных шизофренией более низкие по сравнению с уровнями у здоровых индивидов, достигая статистически значимой разницы (t = 2.72, p = 0.009). Пациенты женского пола показывают более низкие уровни BDNF по сравнению с мужчинами, но разница не достигает статистической значимости (t = 0.1, p = 0.9). ЗАКЛЮЧЕНИЕ: Редукция в сывороточных уровнях BDNF предполагает возможный дефицит в продукции этого нейротрофического фактора и подкрепляет идею, что BDNF можно связать с шизофренией

Chronic treatment with the new anticonvulsant drug lacosamide impairs learning and memory processes in rats: A possible role of BDNF/TrkB ligand receptor system

ABSTRACT Cognitive impairment is considered a frequent side effect in the drug treatment of epilepsy. The objective of the present study was to investigate the effects of lacosamide (LCM) on learning and memory processes in rats, on the serum level of brain‐derived neurotrophic factor (BDNF) and BDNF/TrkB ligand receptor system expression in the hippocampal formation. Male Wistar rats underwent long‐term treatment with three different doses of lacosamide – 3 mg/kg (LCM 3), 10 mg/kg (LCM 10) and 30 mg/kg (LCM 30). All rats were subjected to one active and one passive avoidance tests. The BDNF/TrkB immunohistochemical expression in the hippocampus was measured and serum BDNF was determined. The LCM‐treated rats made fewer avoidance responses than controls during acquisition training and in the memory retention test. The number of escapes in the LCM 10 and LCM 30 groups decreased throughout the test, while the rats in the LCM 3 group showed fewer escapes only in the memory test in the active avoidance task. In the step‐down test, the latency time of the LCM‐30 treated rats was reduced as compared with the controls during the learning session and the short‐ and long‐term memory retention tests. Lacosamide induced a dose‐dependent reduction of the hippocampal expression of BDNF and its receptor TrkB. We found no significant difference between BDNF serum levels in the test animals and controls. The results of the study suggest that LCM suppresses the learning and memory processes in rats, with the inhibition of hippocampal BDNF/TrkB ligand receptor system being one of the possible mechanisms causing this effect. HighlightsLCM dose‐dependently impairs cognition in the active avoidance task.The drug dose of 30 mg impairs learning in the step‐down task.LCM impairs short‐ and long‐term memory in the step‐down test.LCM suppresses the hippocampal expression of BDNF/TrkB ligand receptor system.

Serotonin-Producing Cells in Human Gastric Mucosa - Immunohistochemical and Electron Microscopic Study

Serotonin-Producing Cells in Human Gastric Mucosa - Immunohistochemical and Electron Microscopic Study The great many hormones released by the endocrine cells of the glands and lining epithelium of gastric mucosa determine its significance for the processes in the gastrointestinal tract. One of these hormones, serotonin, plays an important role in the regulation of the motility, secretion and sensation in the gastrointestinal tract. The aim of the present study was to conduct immunohistochemical and electron micro-scopic studies of serotonin-producing EC cell of gastric mucosa. Material and methods: Gastric mucosa biopsies were obtained and studied immunihistochemically for serotonin expression in the mucosa endocrine cells. Electron microscopic study was performed to specify the processes of synthesis, accumulation and release of secretory product by those cells. Results: The immunohistochemical study revealed a considerable number of serotonin-containing EC cells scattered in the lining epithelium and between the glands in the corpus and pyloric region of the stomach. The electron microscopic study followed the stages of formation of the secretory granules from the initial accumulation of granular substance, its membrane packing and formation of mature granules to their disintegration in the secretory process. Conclusions: Serotonin as a neurotransmitter and gastrointestinal hormone appears to be a key to understanding a number of symptoms of gastrointestinal disorders like nausea, vomiting, pain, diarrhea and constipation. A detailed study of serotonin functions in the gastrointestinal tract realised through different types of receptors, and of the development of specific antagonists and agonists to these receptors would open up new opportunities for a more efficient treatment of gastrointestinal disorders. Серотонин-продуцирующие Клет-ки, Взятые С СЛизистой Оболочки Человека - ИммуногистоХимичес-кое И Электронномикроскопичес-кое Исследования Участие слизистой оболочки в координации про-цессов, протекающиХ в гастроинтестиналъном тракте, определяется большим числом гормонов, секретирующиХся эндокринными клетками в ее железаХ и в покровном эпителии. Один из этиХ гормонов, а именно серотонин, играет важную роль в регуляции мотилитета, секреции и чувственныХ восприятий в гастроинтестиналъном тракте. Цель:Настоящее исследование ставит себе целью провести иммуногистоХимическое и элек-тронномикроскопическое исследования серотонин-продуцирующиХ клеток, взятыХ с слизистой обо-лочки. Материал и методы: Биопсический материал с слизистой- оболочки здоровыХ пациентов иссле-дуется иммуногистоХимически относительно экс-прессии серотонина в ее эндокринныХ железаХ. Электронномикроскопическое исследование изучает в деталяХ процессы синтеза, накопления и секреции секреторного продукта в этиХ клеткаХ. Результаты: ИммуногистоХимическое исследование устанавливает наличие значительного числа серо-тонин-содержащиХ ЭС клеток, разбросанныХ в покровном эпителии и в пилоре желудка. Электрон-номикроскопическое исследование прослеживает этапы при формировании секреторныХ гранул -от первоначального накопления зернистой субстанции, ее упаковки мембраной и формирования зрелыХ гранулдо иХ дезинтеграции при секреторном процессе. Выводы:Серотонин, как нейротрансмиттер и желудочно-кишечный гормон, оказывается клю-чом к ряду симптомов гастроинтестинальныХ заболеваний (тошнота, рвота, боль, диарея, за-пор). Детальное изучение функций серотонина в гастроинтестиналъном тракте, осуществленныХ посредством различныХ типов рецепторов, как и создание специфическиХ антагонистов и агонистов для этиХ рецепторов открывает новые направления в терапии гастроинтестинальныХ заболеваний.

11β Hydroxysteroid Dehydrogenase Type 2 in the Adrenal Gland by Testosterone Withdrawal of Adult Rats

11β Hydroxysteroid Dehydrogenase Type 2 in the Adrenal Gland by Testosterone Withdrawal of Adult Rats Aim: 11β hydroxysteroid dehydrogenase (11β HSD) catalyzes the interconversion of glucocorticoids to inert metabolites in man and rodents and plays a crucial role in regulating corticosteroid hormone action. The physiological role and regulation of 11β HSD type 2 in the adrenal gland remains obscure. Therefore, the aim of the present study was to establish the pattern of 11β HSD type 2 expression in rat adrenal gland under conditions of testosterone withdrawal. Material and Methods: We performed immunohistochemical analyses of adrenal gland sections of ethane dimethanesulphonate (EDS)-treated adult rats. Results: In controls, strong positive 11β HSD type 2 signals were detected in the adrenal cortex cells, but not in the medulla. We observed the lowest 11β HSD type 2 expression intensity 7 days after initial treatment with ethane dimethanesulphonate (EDS) followed by progressive increase in the immunoreactivity toward days 14 and 21. Maximal staining intensity of 11β HSD type 2 in the adrenocorticocytes was found by day 35 after EDS treatment. Conclusions: By using the EDS model the present study provides new data about 11β HSD type 2 expression in the adrenal gland under conditions of testosterone withdrawal of adult rats. Our results elucidate further the functional significance of 11β HSD system in rat adrenal gland and the regulatory role of testosterone in its activity. 11β Гидроксистероид Дегидрогена - 3А Типа 2 В Надпочечнойжелезе ЗрелыХ Крыс В УсловияХ Дефицита Тестостерона Цель: 11 β гидроксистероид дегидрогеназа (11 β ГСД)катализирует превращение глюкокортикостероидое в инертные метаболиты у человека и грызунов и играет важную роль в регуляции действия кортико-стероидныХ гормонов. Физиологическая роль и регу-лирование lift ГСД типа 2 в надпочечной железе все еще остаются неясными. В связи с этим настоящее исследование ставит себе целью установить модель экспрессии lift ГСД типа 2в надпочечной железе крыс в условияХ дефицита тестостерона. Материал и методы:Применен иммуногисто- Химический анализ срезов надпочечной железы зрелыХ крыс. Срезы обработаныэтан диметансульфонатом (ЭДС). Результаты: У крыс контрольной группы llβ ГСД типа 2сигналы сильной интенсивности обна-ружены в клеткаХ надпочечной коры, но не и в медулле. Самая слабо выраженная 11 β ГСД типа 2 экспрессия установлена 7 дней после обработки с помощью ЭДС; следует прогрессивное увеличивание интенситета иммунореактивности на 14-ый и 21-ый день. Максимальный интенситет окрашивания 11 р ГСД типа 2 в адренокортикоцитаХ обнаружен на 35-ый день после ЭДС воздействия. Выводы: Применяя ЭДС модель, настоящее иссле- дование дает новые данные насчет экспрессии ll β ГСД типа 2в надпочечнике зрелыХ крыс в условияХ дефицита тестостерона. Полученные результаты способствуют выяснению функционального значения 11 β ГСД системы в надпочечной железе, а также и выяснению регуляторной роли тестостерона в ее активности.

Neurotrophic Factor Receptors trkB and trkC in Experimental Model of Lesion in Rat Brain Structures in Schizophrenia.

Abstract INTRODUCTION: The maldevelopmental model of schizophrenia postulates pathological alterations in embryonal neurogenesis as the etiopathogenetic basis of schizophrenic psychoses. The neurotrophic factor hypothesis explains these neuropathological abnormalities as the result of alterations of the neurotrophin system caused by different mechanisms such as a genetic, infectious and traumatic factors. The tyrosine-kinase containing receptors trkB and trkC mediate growth-promoting effects of neurotrophins and respond to changes in neurotrophic factors availability. AIM: The aim of the present study was to establish the expression pattern of trkB and trkC in rat brain structures by a developmental model of schizophrenia. MATERIALS AND METHODS: On cryostat coronal brain sections of control and lesioned rats (after infusion of ibotenic acid solution bilaterally into the hippocampal formation), immunoreactions for trkB and trkC were performed. RESULTS: We found diminished expression of trkB and trkC in the hippocampal formation of lesioned animals compared to the controls. Quantitative measurements of immunohistochemical reactions intensity and statistical analysis confi rmed the reduced immunoreactivity for antigens under study (trkB and trkC) in the positive hippocampal neurons of 56-day-old lesioned rats compared to the control animals. CONCLUSION: The observed downregulation of neurotrophic factor receptors expression may compromise the function and plasticity of hippocampal formation in schizophrenic brains. Резюме ВВЕДЕНИЕ: Модель шизофрении, связанной с нарушениями в развитии постулирует патологические отклонения в эмбриональном нейрогенезе в качестве этиопатогенетической основы шизофренических психозов. Гипотеза о нейротрофических факторах объясняет данные нейропатологические отклонения как результат нарушений в системе нейротрофинов, вызванных различными генетическими, инфекциозными и травматическими факторами. Тирозинкиназные рецепторы trkB и trkC опосредуют эффекты нейротрофинов, стимулирующих рост и соответствуют изменениям в их наличности. ЦЕЛЬ: Целью настоящего исследования является установление модели экспрессии trkB и trkC в мозговых структурах крысы при экспериментальной модели шизофрении. МАТЕРИАЛЫ И МЕТОДЫ: На криостатных срезах мозга контрольных и испытуемых крыс (после билатеральной инфузии изибутановой кислоты в гиппокампальную формацию) была прослежена иммунореактивность на trkB и trkC. РЕЗУЛЬТАТЫ: Наш анализ выявил заниженную экспрессию рецепторов trkB и trkC в гиппокампальной формации испытуемых животных по сравнению с контрольными. Количественное измерение интенсивности иммуногистохимических реакций и статистический анализ подтвердили понижение иммунореактивности исследуемых антигенов (trkB и trkC) в позитивных гиппокампальных нейронах у испытуемых крыс 56-дневного возраста по сравнению с иммунореактивностью контрольных животных. ЗАКЛЮЧЕНИЕ: Установленное понижение в экспрессии рецепторов нейротрофических факторов можно рассматривать в связи с нарушением функции и пластичности гиппокампальной формации в мозге при шизофрении.

Morphological Changes in Rat Leydig Cells Reflecting the Decreased Testicular Steroidogenic Capacity during Aging

The Leydig cells situated in the interstitial compartment of the mammalian testis are responsible for most of the testosterone produced in males. Previous studies in rats have shown that the ability of Leydig cells to produce testosterone significantly declines with age. The present study was focused on the description of some morphological alterations in rat Leydig cells that could be associated with the decreased testicular steroidogenic capacity during aging. Ultrastructural study of aging rat testes revealed the presence of Leydig cells with intact morphology as well as Leydig cells with different degree of degeneration. The decrease of both smooth endoplasmic reticulum and mitochondria, together with an accumulation of lipid droplets and residual bodies in aging Leydig cells, were observed. Our electron microscopical analysis revealed that the majority of the mitochondria in aged Leydig cells (24-month-old) exhibited significant features of degeneration: increasing in size, swelling, lighten of the matrix, reduction of cristae number, and alterations in the structural integrity of the membranes. During aging progressive increase was established in the number of Leydig cells exhibiting nuclear changes such as chromatin compaction and fragmentation and nuclear shrinkage that could be a sign of elevated apoptotic tendency. The observed morphological alterations in rat Leydig cells reflect the decreased testicular steroidogenic capacity during aging, resulting in decrease of testosterone production.

Hydrohysteroid Dehydrogenases – Biological Role and Clinical Importance – Review

Hydroxysteroid dehydrogenases (HSDs) belong to the NADPH/NAD+-dependent oxidoreductases, which interconvert ketones and the corresponding secondary alcohols. As their names imply, they catalyze the oxidoreduction in different positions of steroidal substrates (3α-, 3β-, 11β-, 17β-, 20αand 20β-position). The steroid-converting HSDs play central roles in the biosynthesis and inactivation of steroid hormones, but some of them are also involved in the metabolism of diverse non-steroidal compounds [1]. The HSDs are integral parts of systemic (endocrine) and local (intracrine) mechanisms. In target tissues they convert inactive steroid hormones to their corresponding active forms and viceversa, thus modulating the transactivation of steroid hormone receptors or other elements of the non-genomic signal transduction pathways. Therefore, HSDs act as molecular switches allowing pre-receptor modulation of steroid hormone action [2].