Contribution au mécanisme d'induction de la castration médicale avec les agonistes de la LHRH chez l'homme et au mécanisme d'action de l'EGF chez le rat

La première partie de cette thèse est consacrée à la caractérisation des récepteurs ovariens et prostatiques pour l''epidermal growth factor (EGF) et à l'étude des événements moléculaires suivant la liaison de l'EGF à son récepteur dans l'ovaire chez le rat. En utilisant l'EGF purifié marqué à l'iode⁻¹²⁵, nous avons étudié le site récepteur pour l'EGF dans le lobe ventral de la prostate de rat. Ce récepteur montre une haute affinité pour son ligand (KD = 0.93 ± 0.09 nM). La concentration des récepteurs EGF prostatiques est sous contrôle androgénique: la castration chirurgicale ou chimique (avec un agoniste de la LHRH combiné è un antiandrogène) stimule le niveau des récepteurs alors que la dihydrotestostêrone produit l'effet inverse. Par la suite, nous avons mis en évidence un site récepteur spécifique de haute affinité (KD = 1.1 ± 0.2 nM) pour l'EGF dans l'homogénat total d'ovaire. Le niveau de ces récepteurs EGF ovariens varie en fonction du cycle estrien du rat, les niveaux les plus bas étant observés aux jours diestrus-1 et diestrus-2 et la capacité de liaison de l’EGF dans l'ovaire augmentant en après-midi du jour proestrus pour se maintenir élevée en estrus. Ces variations du niveau des récepteurs EGF ovariens au cours du cycle estrien pourraient être causées par les gonadotrophines puisque nous avons démontré que l'administration chronique de gonadotrophines exogènes (hCG et FSH) stimule le niveau des récepteurs EGF dans l'ovaire. Les androgènes inhibent légèrement les concentrations de récepteurs pour l'EGF dans l'ovaire. En utilisant les techniques de radioautographie, nous avons localisé le récepteur EGF sur les cellules de la granulosa, les cellules de la thèque interne et les cellules lutéales dans l'ovaire de rat. Lorsque l'EGF se lie à son récepteur sur les membranes des cellules ovariennes, il stimule la phosphorylation d'une composante membranaire de Mr = 37K. Cette stimulation est rapide et spécifique à l'EGF. Dans des membranes isolées de foie de rat, l'EGF stimule la phosphorylation d'une protéine de Mr = 160K. Nos travaux démontrent l'existence de sites récepteurs spécifiques pour l'EGF dans la prostate et l'ovaire de rat et suggèrent que la stimulation de la phosphorylation d'une protéine de Mr = 37K pourrait être une étape importante dans le mécanisme d'action spécifique du peptide dans l'ovaire.Dans la deuxième partie, nous avons déterminé l'effet d'un traitement chronique avec un agoniste de la LHRH sur l’activité biologique de la LH chez l'homme. Nous avons utilisé comme modèle les patients atteints de cancer de la prostate et traités par la nouvelle thérapie combinée utilisant un agoniste de la LHRH, et un antiandrogène, mise au point dans notre laboratoire. Un système in vitro très sensible utilisant la sécrétion de testostérone par une suspension de cellules de Leydig de souris a été utilisé pour évaluer l'activité biologique de la LH dans le sérum des patients. Nos résultats démontrent que l'activité biologique de la LH est sélectivement inhibée après 1 mois de thérapie combinée alors que les niveaux de LH mesurés par radioimmunoétalonnage (RIA) ne sont que peu affectés. L'administration chronique d'un agoniste de la LHRH en combinaison avec un antiandrogène provoque aussi une désensibilisation hypophysaire, c'est-à-dire une inhibition progressive de la réponse en LH stimulée par la LHRH. Chez des patients castrés recevant seulement l'antiandrogène, l'activité biologique de la LH n'est pas affectée, suggérant que la LHRH, et non l'antiandrogène, provoque la perte de bioactivité de l'hormone lors du traitement combiné. Après avoir caractérisé deux radioimmunoétalonnages pour les sous-unités α et β de la LH, nous avons démontré que la thérapie combinée stimule les concentrations sériques de la sous-unité a chez les patients atteints de cancer de la prostate, sans affecter les niveaux de la sous-unité β. La réaction croisée des niveaux stimulés de sous-unité a dans le RIA de la LH n'est pas suffisante pour expliquer les niveaux de LH immunoréactive mesurés lors du traitement combiné démontrant que la perte de bioactivité de la LH induite par la thérapie combinée n'est pas due à la dissociation complète de l'hormone en ses sous-unités constituantes.Chez le chien, l'administration chronique d'un agoniste de la LHRH provoque une stimulation transitoire des niveaux de LH bioactive et de testostérone suivie d'une inhibition quasi-totale des concentrations de ces deux hormones. L'administration concomitante d'inhibiteurs de la stéroïdogénèse empêche l'élévation initiale des concentrations de testostérone sans modifier de façon appréciable le profil de sécrétion de la LH bioactive. Ces résultats démontrent que l'administration chronique d'un agoniste de la LHRH en combinaison avec un antiandrogène pur chez des patients atteints de cancer de la prostate stimule les niveaux sériques de la sous-unité a de la LH et inhibe de façon importante l'activité biologique de la LH circulante. Le parallélisme étroit existant entre l’inhibition des concentrations circulantes de testostérone et la perte de bioactivité de la gonadotrophine suggère que cette inhibition de l'activité biologique de l'hormone est responsable de la castration médicale provoquée par la thérapie combinée. Le chien pourrait être utilisé comme modèle afin de déterminer les modifications de la molécule de LH provoquée par l'agoniste de la LHRH et conduisant à la réduction de l'activité biologique de la gonadotrophine.Montréal Trigonix inc. 201

Steroids

Steroids: The basic science and clinical aspects covers the modern understanding and clinical use of steroids. The history of steroids is richly immersed and runs long and deep. The modern history of steroids started in the early 20th century, but its use has been traced back to ancient Greece. We start by describing the basic science of steroids. We then describe different clinical situations where steroids play an important role. We hope that this book will contribute further to the literature available about steroids and enables the reader to further understand this interesting and rapidly evolving science

Chronic Use of Deslorelin in Dogs: Six Cases (2005–2022)

openIl deslorelin, un agonista dell’ormone rilasciante gonadotropine, è attualmente registrato per l'induzione dell'infertilità temporanea nei cani maschi, nei gatti maschi, nei furetti maschi e anche nelle cagne prepuberi, ma vari studi hanno dimostrato la sua utilità anche per altre condizioni che richiedono un trattamento cronico. L'efficacia e la sicurezza, nonché il ritorno alla fertilità di questo metodo di sterilizzazione non chirurgica sono ben noti dopo un singolo utilizzo, ma i dati disponibili sul suo uso prolungato sono molto limitati. Per dare ulteriore contributo all’uso cronico di questo farmaco, abbiamo pubblicato un articolo scientifico che presenta sei casi di cani trattati cronicamente con deslorelin tra il 2009 e il 2022. I cani presentati in questo lavoro sono stati trattati per 2-9 anni consecutivi a scopo anticoncezionale (un caso) e per condizioni non riportate sul foglietto illustrativo del farmaco: iperplasia prostatica benigna e malattia della ghiandola perineale (un caso), controllo del comportamento riproduttivo nei cani maschi (due casi) e incontinenza urinaria in cagne adulte sterilizzate (due casi). Tutti gli animali erano in buona salute durante il trattamento e non hanno presentato effetti collaterali a breve termine. Reazioni di flare-up (aumento del comportamento riproduttivo per 1-3 settimane dopo il trattamento) sono state osservate in 1/4 dei maschi interi e non sono state osservate nelle cagne incontinenti sterilizzate. Il deslorelin è stato efficace in tutti i cani trattati. La fertilità è stata immediatamente recuperata in un cane maschio che ha prodotto una cucciolata quando il proprietario ha tardato l’inserimento di un nuovo trattamento, necessario per mantenere e prolungare l'effetto. Due cani hanno sviluppato una neoplasia: una cagna sterilizzata trattata per 3.5 anni per incontinenza urinaria ha sviluppato un carcinoma ipofisario e un cane maschio intero trattato per 9 anni per il controllo della fertilità ha sviluppato un carcinoma vescicale. Entrambe le neoplasie non risultano avere alcun rapporto con il trattamento a base di deslorelin. Il valore scientifico di questo lavoro è costituito da case reports su nuove applicazioni e l'uso cronico del deslorelin. Dopo quasi 20 anni di utilizzo del farmaco, è essenziale riportarne tutti gli effetti sospettati o confermati, poiché questi dati son fondamentali per l'uso sicuro del farmaco e per problemi legati alla gonadectomia e le sue alternative. Questo lavoro conclude che gli impianti di deslorelin possono essere considerati un'alternativa sicura alla castrazione chirurgica in patologie specifiche mediate dagli ormoni riproduttivi e in situazioni in cui la castrazione chirurgica non è un'opzione, come nel caso di animali affetti da condizioni cardiovascolari o altre malattie sistemiche che rendono l'anestesia non sicura. Nonostante la limitata numerosità campionaria, possiamo affermare che il trattamento cronico con deslorelin è considerato presumibilmente sicuro quando l'animale viene trattato per un tempo illimitato e, potenzialmente, a vita, ma che sono necessarie ulteriori ricerche per confermare questa ipotesi.Deslorelin, a long-acting gonadotropin-releasing hormone agonist, is currently registered for the induction of temporary infertility in male dogs, male cats, male ferrets, and also prepubertal female dogs, but research has shown its usefulness for other conditions requiring chronic treatment. Efficacy and safety as well as return to fertility of such non-surgical neutering methods are well known following a single use but little if any data is available on prolonged use. To offer further support to this claim, we published a paper presenting six cases of dogs chronically treated with deslorelin between 2009 and 2022. The dogs presented in this paper were treated for 2 to 9 consecutive years for ensuring failure to reproduce (one case) as well as for conditions which are not found on the drug leaflet: benign prostatic hyperplasia and perineal gland disease (one case), control of reproductive behavior in male dogs (two cases) and urinary incontinence in spayed adult bitches (two cases). All animals were in good health during treatment and presented no short-term side effects. Flare-up reactions (an increase in reproductive behavior for 1–3 weeks after treatment) were observed in 1/4 intact males and were not observed in the spayed incontinent bitches. Deslorelin was effective in all treated dogs. Fertility was immediately regained in one male dog who sired a litter when his owner forgot to come back for re-treatment at the right time to maintain and prolong its effect. Two dogs developed a neoplasia: a spayed bitch treated for 3.5 years for urinary incontinence developed a pituitary carcinoma, and an intact male dog treated for 9 years for the control of fertility developed a bladder carcinoma. Both neoplasia were considered unrelated to the treatment. This paper provides valuable clinical information on new applications and chronic use of deslorelin. After almost 20 years of using the drug, it is essential to report all suspected or confirmed effects as these data are critical for the safe use of the drug and for the problems related to gonadectomy and its alternatives. Based on this work, deslorelin implants can be considered as a safe alternative to surgical castration in specific pathologies mediated by reproductive hormones and in situations where surgical castration is not an option, such as animals suffering from cardiovascular conditions or other systemic diseases which make anesthesia unsafe. Despite the limited sample size, we can state that chronic treatment with deslorelin is considered potentially safe when an animal is being treated for an unlimited time period and, potentially, for life, but that further research is necessary to investigate and confirm this hypothesis

Proliferative responses of normal rat ventral prostate 'in vitro'

Proliferative responses of normal rat ventral prostate in vitro were investigated using the incorporation of 5-[125I]-iodo-2'-deoxyuridine (125I-UdR) to monitor DNA synthesis, with the aim of establishing a model system for evaluating the direct effects of hormones and chemotherapeutic agents on prostatic growth. In androgen-free, chemically-defined organ culture, the proliferative activity of young adult rat (4 to 6 months old) ventral prostate declined with time and the tissue underwent retrogressive changes resembling post-castration atrophy in vivo. Treatment with testosterone (4 x 10-12 to 4 x 10-5M) exhibited a dose-dependent response, with concentrations ranging from 4 x 10-9to 4 x 10-6M preventing the retrogressive changes associated with androgen deprivation and eliciting maximal increases (approximately 3-fold) in 125I-Udr uptake. Higher testosterone concentrations (2.5 x 10-5 to 4 x 10-5M) exerted a non-specific cytotoxic effect, resulting in marked suppression of 125I-Udr uptake. Following stimulation with an optimal concentration of testosterone (4 x 10-7M), 125I-Udr incorporation reached peak activity on day 4 of the culture period and rapidly declined thereafter, despite the continued presence of testosterone. Effects of variations in organ culture media and methodology were also examined to further establish optimal conditions for investigations of the proliferative response to testosterone stimulation. The established method of quantitative organ culture was then used to compare the proliferative effect of testosterone with that of its major metabolites, 5α-dihydrotestosterone, androstenedione, androstenedione and 5α-androstane-3β, 17β-diol

Studies of the physiology and biochemistry of the male reproductive tract.

In order to develop an effective contraceptive for the male, if is necessary to fully understand the anatomy, physiology and biochemistry of the male reproductive tract, and this is reviewed in Chapter I

Direct actions of a gonadotropin-releasing hormone agonist on steroidogenesis and aromatase activity in cultured porcine granulosa cells

The mechanism by which a gonadotropin-releasing hormone agonist (D-Ala6) des-Gly10-GnRH (GnRHa) modulates ovarian steroid secretion was investigated in cultured porcine granulosa cells. Ovarian granulosa cells, obtained from medium (3-5 mm) sized follicles, were incubated with various hormones in vitro and the steroid content of the spent medium determined by radioimmunoassay. The morphology of the granulosa cells were initially epithelioid, but tended to assume a fibroblast-like morphology during the course of the culture. In comparison with control cultures, it was determined that the hormone had no systemic effect on morphology or cell growth in culture. Graded doses of hFSH or 8-Br-cAMP caused significant increases in progesterone secretion, but had little effect on estradiol secre-tion. Granulosa cell response to GnRHa was generally dose-dependent, with GnRHa causing increases in both progesterone and estrogen secretion. The GnRHa-induced actions were blocked in a dose-dependent manner by a GnRH antagonist Ac-D-Ala1, D-Phe2, D-Trp3,6 GnRH. GnRHa (1 µg) inhibited FSH- (100 ng/ml) and 8-Br-cAMP- (0.1 mM) stimulated progesterone secretion. Moreover, GnRHa stimulated basal progesterone secretion in untreated cells or in cells treated with only hCG (1 µg/ml). However, GnRHa stimulated estradiol secretion in untreated, FSH-treated, as well as hCG-treated granulosa cells. This secretion, however did not occur in cells treated with 8-Br-cAMP. GnRHa decreased progesterone secretion at the same time it increased estradiol secretion in granulosa cells isolated from large (6-12 mm) follicles. To further examine the GnRHa-induced increase in estrogen secretion, an aroma-tase assay was performed on both untreated and GnRHa treated cells. GnRHa stimulated aromatase activity over 4 d of continuous incuba-tion. Removal of GnRHa from the medium resulted in aromatase activ-ity returning to control levels. This study demonstrated that GnRHa has both stimulatory and inhibitory actions on steroidogenesis in the cultured porcine granu-losa cell. GnRHa decreases of FSH- and 8-Br-cAMP stimulated pro-gesterone secretion suggests that some of the GnRHa actions lie distal to cAMP formation. One of these actions demonstrated by GnRHa stimulation of estrogen secretion, with or without gonado-tropins, and subsequent aromatase assay is GnRHa-induced increase in aromatase activity. Furthermore, continuous exposure to GnRHa is necessary to elicit the increased aromatase activity

The Role of Androgens in Testicular Development and Dysgenesis

Disorders of male reproductive health which manifest at birth (cryptorchidism, hypospadias) or in young adulthood (testicular germ cell cancer and low sperm counts), are common and may be increasing in incidence. These disorders have a common fetal origin and share risk factors; consequently they are hypothesized to comprise a testicular dysgenesis syndrome (TDS). TDS arises when maldevelopment (dysgenesis) of the fetal testis results in hormonal malfunctions and abnormal development and function of the somatic cells. It is thought that the suppressed intratesticular testosterone levels associated with TDS may account for subsequent low sperm counts, via a reduction in perinatal Sertoli cell proliferation/number. Sertoli cells do not express androgen receptors (AR) in fetal life in the human or rat, so it is hypothesised that any androgen effects on Sertoli cell number occur indirectly, via the AR positive peritubular myoid cells. Evidence from the di (n‐butyl) phthalate (DBP)‐treated rat model for TDS suggests that reduced androgen action may play a role in testicular dysgenesis as in patients with complete androgen insensitivity syndrome (CAIS; ‘testicular feminization’), in whom focal areas of testicular dysgenesis have been reported. The studies in this thesis sought to establish if reduced androgen levels/action in the fetal rat testis contribute to putative testicular dysgenetic features, namely reduced Sertoli cell number, occurrence of multinucleated gonocytes or abnormal aggregation of fetal Leydig cells, the precursor of focal dysgenesis. Pregnant rats were exposed to treatments or co‐treatments expected to manipulate testicular testosterone levels (DBP, testosterone propionate; TP) or action (flutamide, DMBA) or to induce intrauterine growth restriction (dexamethasone), another risk factor for TDS. The aforementioned endpoints were analysed in fetal testes and related to testicular testosterone levels and peripheral androgen action (anogenital distance). The same endpoints were evaluated in mice with inactivation of the androgen receptor (tfm or ARKO mice). As androgen action is assumed to be mediated indirectly, via the peritubular myoid cells, changes in peritubular myoid cell number and function were investigated in testes with suppressed androgens. In vitro studies were also used to investigate the role of androgens in Sertoli cell proliferation. Fetal rat testis explants were cultured with various chemicals designed to manipulate androgen action and Sertoli cell proliferation. Potential non‐androgen related mechanisms of DBP action were investigated using Taqman RT‐PCR to determine the mRNA expression of key developmental genes after exposure to DBP. Sertoli cell number was reduced after exposure to treatments that reduced testicular testosterone levels, i.e. DBP alone or as a co‐treatment, TP and dexamethasone. Sertoli cell numbers in ARKO mice were also significantly reduced. The occurrence of multinucleated gonocytes and large Leydig cell clusters were induced after exposure to DBP, alone or as a co‐treatment, but not after exposure to TP or dexamethasone, and these dysgenetic endpoints did not occur either in tfm or ARKO mice. Rats exposed in utero to DBP have reduced testicular testosterone levels, however peritubular myoid cell number was unaffected by DBP, though AR expression in the peritubular myoid cells was delayed, and laminin and vimentin expression in Sertoli cells was altered after DBP exposure. DMRT‐1 and DAX‐1 mRNA expression levels were significantly reduced after DBP exposure, but this reduction was no longer evident once mRNA expression was corrected for Sertoli cell number. In conclusion, these studies provide strong evidence that androgens play a role in regulation of Sertoli cell number/proliferation, and this is supported by a comparable reduction in Sertoli cell number in ARKO and tfm mice. However, since the treatments that reduce testicular testosterone in the rat, may also have a direct affect on the Sertoli cells, this alternate mechanism of action cannot be ruled out, and the administration of a treatment that reduces testicular testosterone without directly affecting Sertoli cells is required. These studies also show that reduced testicular testosterone levels are associated with multinucleated gonocyte formation and fetal Leydig cell aggregation, although this evidence it is not supported by parallel findings from the TP and dexamethasone exposed rats or the ARKO and tfm mice, as neither of these endpoints were identified as being affected in these animals. Aside from the delay in AR expression, there were no obvious changes in peritubular myoid cell number or the peritubular myoid cell markers examined in testes deprived of androgens, although there are other markers that could be investigated. mRNA analysis of the developmental genes investigated after DBP exposure, demonstrated no change in expression after correction for Sertoli cell number, suggesting that they do not play a role in the dysgenetic features observed in DBP exposed testes

Testicular factors involved in testis descent

Testis descent is the male-specific developmental process by which the gonads descend from their original position in the abdomen to the final position in the scrotum. Scrotal positioning of the testes, as it occurs in many mammals, is essential for spermatogenesis. Since the time of John Hunter in 1762, many scientists have studies testis descent, which turned out to be a complex process, involving an interplay of different structures and factors. Due to this complexity, the understanding of the structural and molecular mechanisms underlying the process of testis descent is still far from complete