Molekularbiologische Untersuchung des CAG-Repeats des humanen Androgenrezeptors und Korrelation mit der Histologie der Spermatogenese - Ist das CAG-Repeat ein molekularer Marker für männliche Infertilität?

In der vorliegenden Doktorarbeit sollte geklärt werden, inwieweit ein Zusammenhang zwischen dem polymorphen CAG-Repeat des humanen Androgenrezeptors (AR) und a) der Histologie der ungestörten und auch gestörten Spermatogenese, b) der AR- und ABP-Expression selbst und c) der Testosteronkonzentration im Blut besteht. Das CAG-Repeat ist bekannt durch die spino-bulbäre Muskelatrophie (SBMA, Kennedy’s Disease), die neben den neuro-muskulären Degenerationen mit einer Beeinträchtigung der Fruchtbarkeit einhergeht. Bei dieser Erkrankung ist zudem das Vorkommen von Mosaiken bekannt, d.h. das Auftreten unterschiedlich langer CAG-Repeats im AR-Gen verschiedener Gewebe. Es stellte sich die Frage, ob auch ein moderat verlängertes, aber immer noch innerhalb der physiologischen Grenzen liegendes CAG-Repeat Einfluss auf die männliche Fertilität nehmen kann. Darüber hinaus war fraglich, ob die Bestimmung des Trinukleotidrepeats aus dem Blut als diagnostisches Werkzeug verwendet werden kann, oder ob zwischen den Kompartimenten Blut und Hoden bzw. innerhalb der Zellpopulationen im Hoden selbst ein somatisches Mosaik besteht. Deswegen untersuchten wir das CAG-Repeat nicht nur in der DNA aus Blutlymphozyten, sondern auch in den Zellpopulationen des Hodens auf DNA- und mRNA-Ebene. Einzelne Zellpopulationen konnten durch die Technik des Laser-assistierten Zellpickings (LACP) isoliert voneinander gewonnen und untersucht werden. Verwendet wurden zwei verschiedene Techniken zur Bestimmung des CAG-Repeats. Zum wurde die Fragmentlängenanalyse als Standardmethode durchgeführt, zum anderen die in diesem Zusammenhang neue native Polyacrylamidgel-Elektrophorese (PAGE). Wir konnten Folgendes mit unseren Untersuchungen zeigen: a) alle von uns bestimmten Repeats waren innerhalb der physiologischen Grenzen b) es gibt keinen Zusammenhang zwischen dem CAG-Repeat und dem histologisch definierten Spermatogenesestatus c) die AR-Expression im Hoden ist unabhängig von Spermatogenesestörungen und CAG-Repeatlänge d) die ABP-Expression ist bei Spermatogenesestörungen signifikant herunterreguliert, aber ebenfalls unabhängig von der CAG-Repeatlänge e) die CAG-Repeatbestimmung innerhalb der physiologischen Grenzen ist anfällig für Störungen der Polymeraseaktivität durch Bildung von DNA-Sekundärstrukturen f) bei Patienten mit einer bunten Atrophie der Spermatogenese können Unterschiede in der CAG-Repeatlänge zwischen Sertolizellen auftreten, die aus Tubuli mit normaler und gestörter Spermatogenese stammen Störungen in der Replikation und Transkription können durch Wasserstoffbrückenbindungen zwischen den Basen Guanin und Cytosin bei CAG-Repeats leicht entstehen und sind als Ursache von Replikationsabbrüchen bei Bakterien und Hefen schon lange bekannt. Weitere Untersuchungen in der Arbeitsgruppe sollen klären, inwieweit auch im physiologischen Rahmen liegende CAG-Repeats die Expression von Zielgenen des AR beeinflussen können und ob es eine optimale CAG-Repeatlänge für die AR- Funktion gibt.It was the aim of this doctoral thesis to elucidate, whether there is a connection between the polymorphic CAG repeat of the human androgen receptor (AR) and a) normal and impaired spermatogenesis, b) AR and ABP expression itself and c) testosterone concentration in the peripheral blood. The CAG repeat has gained interest in the context of spinal bulbar muscular atrophy (SBMA, Kennedy’s disease), which is not only characterized by neuromuscular degeneration but also impairment of fertility in men. In SBMA, mosaics are known to occure, meaning that distinct CAG repeat lengths in the AR gene in different tissues exist. It is in question, whether also moderate expanded but still physiological CAG repeats may influence spermatogenesis. At the same time it is questionable, if the determination of CAG repeat length in peripheral blood is a useful diagnostic tool for spermatogenic impairment detection or if a somatic mosaicism between blood and testis in within the testis itself exists. For this purpose, we were not only examining CAG repeat in blood lymphocyte DNA, but also in different cell populations of the testis on DNA and mRNA level. Single cell populations were assessed separately by applying laser assisted cell picking (LACP) technique and examined independently from each other. We used two different methods for CAG length determination, fragment length analysis as standard method on the one, and additionally native polyacrylamide gel electrophoresis on the other hand. We were able to show the following results: a) all assessed CAG repeats were within the physiological range b) there is no correlation between CAG repeat length and histologically assessed status of spermatogenesis c) the AR expression is independent of CAG repeat length and impairment of spermatogenesis d) the ABP expression is significantly down regulated in patients with severely impaired spermatogenesis but independent of CAG repeat length e) determination of CAG repeat length within the physiological range is susceptible for polymerase failure due to the formation of secondary DNA structures f) patients showing a mixed atrophy of spermatogenesis show CAG length differences in Sertoli cell populations originating from tubules showing normal or impaired spermatogenesis Defects of replication and transcription machinery may be caused by H+ bondings between the bases Guanin and Cytosin, which develop easily and are known to stop replication processes in bacteria and yeast. Further experiments in this working group have to elucidate, whether even CAG repeats within the physiological range can influence gene expression of AR target genes and if an ideal CAG repeat length for AR function exists

Task force veterinary anatomy: Joint efforts of the five German veterinary schools to ensure education during the COVID-19 pandemic

At the start of the COVID-pandemic in March 2020, the Institutes of Veterinary Anatomy of the five German educational institutions were confronted with the challenge of digitalising all lectures for the second and fourth semesters of veterinary students. After an online kick-off event and a preliminary status quo meeting, all available digital teaching material was exchanged for students to stream from learning platforms. Lectures were either synchronized or made available as audio recordings and connotated slides on the learning platforms. Fortunately, digital microscopic slides had already been in use, which made it easy for students to access them. Dissection exercises mostly consisted of self- study, using instructive videos and interactive exercises. In the second half of the semester, four of the educational institutions were able offer a restricted number of in-person gross anatomy classes under reinforced conditions. Success monitoring took place online through different formats, and partially on a voluntary basis, via the learning platforms. Although the past two semesters had to almost exclusively take place online due to the unprecedented circumstances, and joint efforts of the five veterinary institutions, there is a general consensus that the practical education in anatomy, histology and embryology is essential to veterinary students. In fact, it is the only way they can obtain the necessary skills to successfully complete the rest of their degree.Die Veterinäranatomischen Institute der fünf deutschen Bildungsstätten standen zu Beginn der Coronapandemie im März 2020 vor der Herausforderung, die gesamte Lehre für die Studierenden des zweiten und vierten Fachsemesters in digitaler Form durchzuführen. Nach einem Kickoff-Onlinemeeting und einer ersten Bestandsaufnahme wurden vorhandene digitale Lehrmedien zum Streamen auf den Lernplattformen ausgetauscht. Vorlesungen wurden synchron gehalten oder asynchron als vertonte bzw. annotierte Dateien auf den Lernplattformen hochgeladen. Die mikroskopischen Präparate konnten den Studierenden dank schon bestehender Anwendungen zur virtuellen Mikroskopie komplikationslos zur Verfügung gestellt werden. Die Präparierübungen wurden überwiegend zum Selbststudium anhand von Lehrvideos und interaktiven Übungsaufgaben angeboten. In der zweiten Semesterhälfte wurden in der makroskopischen Anatomie an vier Standorten einige wenige Präsenztermine für die Studierenden unter verschärften Hygienebedingungen angeboten. Erfolgskontrollen wurden ausschließlich online über die Lernplattformen in verschiedenen Formaten und zum Teil fakultativ durchgeführt. Auch wenn die vergangenen beiden Semester in Zusammenarbeit aller Standorte den Umständen geschuldet fast ausschließlich digital stattfinden musste, besteht der allgemeine Konsens, dass die praktische Ausbildung in den Fächern Anatomie, Histologie und Embryologie essentiell für die Studierenden der Veterinärmedizin ist. Nur so können sie die notwendigen Kenntnisse und Fertigkeiten für ihr weiteres erfolgreiches Studium erlangen

Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation

Background: Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR. Results: Transfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes. Conclusion: We conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself

Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies

Background: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. Methods: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines’ representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. Results: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. Conclusion: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression

Modelling the functions of Polo-like kinases in mice and their applications as cancer targets with a special focus on ovarian cancer

Polo-like kinases (PLKs) belong to a five-membered family of highly conserved serine/threonine kinases (PLK1-5) that play differentiated and essential roles as key mitotic kinases and cell cycle regulators and with this in proliferation and cellular growth. Besides, evidence is accumulating for complex and vital non-mitotic functions of PLKs. Dysregulation of PLKs is widely associated with tumorigenesis and by this, PLKs have gained increasing significance as attractive targets in cancer with diagnostic, prognostic and therapeutic potential. PLK1 has proved to have strong clinical relevance as it was found to be over-expressed in different cancer types and linked to poor patient prognosis. Targeting the diverse functions of PLKs (tumor suppressor, oncogenic) are currently at the center of numerous investigations in particular with the inhibition of PLK1 and PLK4, respectively in multiple cancer trials. Functions of PLKs and the effects of their inhibition have been extensively studied in cancer cell culture models but information is rare on how these drugs affect benign tissues and organs. As a step further towards clinical application as cancer targets, mouse models therefore play a central role. Modelling PLK function in animal models, e.g., by gene disruption or by treatment with small molecule PLK inhibitors offers promising possibilities to unveil the biological significance of PLKs in cancer maintenance and progression and give important information on PLKs’ applicability as cancer targets. In this review we aim at summarizing the approaches of modelling PLK function in mice so far with a special glimpse on the significance of PLKs in ovarian cancer and of orthotopic cancer models used in this fatal malignancy

Excessive unilateral proliferation of spermatogonia in a patient with non-obstructive azoospermia – adverse effect of clomiphene citrate pre-treatment?

Background!#!Clomiphene citrate has been proposed as pre-treatment for infertile men with non-obstructive, testicular azoospermia (NOA) before surgery for testicular sperm extraction (TESE), especially when serum testosterone is low.!##!Case presentation!#!Here, we report on a 33-year old azoospermic patient with a previous history of repeated 'fresh' TESE and clomiphene citrate therapy (50 mg/day over 6 months) before undergoing microscopically assisted, bilateral testicular biopsy. Comprehensive histological and immunohistochemical work-up revealed a heterogeneous spermatogenic arrest at the level of spermatogonia or primary spermatocytes, with focally preserved spermatogenesis up to elongated spermatids in the right testis. In the left testis, the majority of tubules (> 70%) showed no tubular lumen or regular seminiferous epithelium but a great number of spermatogonia-like cells. These cells proved to be normally differentiated spermatogonia (positive for melanoma associated antigen 4 (MAGEA4), negative for placental alkaline phosphatase (PlAP)) with increased proliferative activity (positive for proliferating cell nuclear antigen (PCNA)) and a slightly higher rate of apoptotic cells. When compared to a tissue control with normal spermatogenesis, expression of sex hormone receptors androgen receptor (AR), estrogen receptor (ER) alpha, and G-protein coupled estrogen receptor 1 (GPER1) was not altered in patient samples. Sertoli cells appeared to be mature (positive for vimentin, negative for cytokeratin 18), whereas the expression of zona occludens protein 1 (ZO-1), claudin 11, and connexin 43 was absent or dislocated in the tubules with abundance of spermatogonia.!##!Conclusion!#!This result suggests that formation of the blood-testis barrier is disturbed in affected tubules. To our knowledge this is the first observation of excessive, non-malignant proliferation of spermatogonia in a NOA patient. Although underlying molecular mechanisms remain to be elucidated, we hypothesize that the unusual pathology was triggered by the high-dose clomiphene citrate treatment preceding testicular biopsy

Task Force Veterinäranatomie: Zusammenarbeit der fünf deutschen Standorte zur Sicherung der Lehre während der COVID-19-Pandemie

At the start of the COVID-pandemic in March 2020, the Institutes of Veterinary Anatomy of the five German educational institutions were confronted with the challenge of digitalising all lectures for the second and fourth semesters of veterinary students. After an online kick-off event and a preliminary status quo meeting, available digital teaching material was exchanged for students to stream from learning platforms. Lectures were either synchronized or made available as audio recordings and connotated slides on the learning platforms. Fortunately, digital microscopic slides had already been in use, which made it easy for students to access them. Dissection exercises mostly consisted of self-study, using instructive videos and interactive exercises. In the second half of the semester, four of the educational institutions were able to offer a restricted number of in-person gross anatomy classes under reinforced conditions. Success monitoring took place online through different formats, and partially on a voluntary basis, via the learning platforms. Although the past two semesters had to almost exclusively take place online due to the unprecedented circumstances, and joint efforts of the five veterinary institutions, there is a general consensus that the practical education in anatomy, histology and embryology is essential to veterinary students. In fact, it is the only way they can obtain the necessary skills to successfully complete the rest of their degree.Die Veterinäranatomischen Institute der fünf deutschen Bildungsstätten standen zu Beginn der Coronapandemie im März 2020 vor der Herausforderung, die gesamte Lehre für die Studierenden des zweiten und vierten Fachsemesters in digitaler Form durchzuführen. Nach einem Kickoff-Onlinemeeting und einer ersten Bestandsaufnahme wurden vorhandene digitale Lehrmedien zum Streamen auf den Lernplattformen ausgetauscht. Vorlesungen wurden synchron gehalten oder asynchron als vertonte bzw. annotierte Dateien auf den Lernplattformen hochgeladen. Die mikroskopischen Präparate konnten den Studierenden dank schon bestehender Anwendungen zur virtuellen Mikroskopie komplikationslos zur Verfügung gestellt werden. Die Präparierübungen wurden überwiegend zum Selbststudium anhand von Lehrvideos und interaktiven Übungsaufgaben angeboten. In der zweiten Semesterhälfte wurden in der makroskopischen Anatomie an vier Standorten einige wenige Präsenztermine für die Studierenden unter verschärften Hygienebedingungen angeboten. Erfolgskontrollen wurden ausschließlich online über die Lernplattformen in verschiedenen Formaten und zum Teil fakultativ durchgeführt. Auch wenn die vergangenen beiden Semester in Zusammenarbeit aller Standorte den Umständen geschuldet fast ausschließlich digital stattfinden musste, besteht der allgemeine Konsens, dass die praktische Ausbildung in den Fächern Anatomie, Histologie und Embryologie essentiell für die Studierenden der Veterinärmedizin ist. Nur so können sie die notwendigen Kenntnisse und Fertigkeiten für ihr weiteres erfolgreiches Studium erlangen

Rescue of p53 functions by in vitro‐transcribed mRNA impedes the growth of high‐grade serous ovarian cancer

Abstract Background The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high‐grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro‐transcribed (IVT) wild‐type (WT) p53‐mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. Methods To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient‐derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR‐8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. Results Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA‐based methods. The introduction of WT p53 mRNA triggered dose‐dependent apoptosis, cell cycle arrest, and potent long‐lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR‐8 cells upon mRNA‐based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double‐strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose‐dependent manner. Conclusions The IVT mRNA‐based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment