Darstellung von Estradiol-17beta- und Progesteronrezeptoren im Corpus luteum der Hündin zu definierten Zeitpunkten im Östrus und Diöstrus

Nachdem weder ein uterines Luteolysin noch ein Mangel an Gonadotropinen (LH, Prolaktin) kausal mit der ab dem ersten Drittel des Diöstrus einsetzenden Luteolyse in Zusammenhang gebracht werden kann, lag vorliegender Arbeit die Arbeitshypothese zugrunde, dass auch beim Hund - analog zur Situation bei anderen Spezies - luteale Steroidhormone als parakrine/autokrine Faktoren eine Rolle bei der Steuerung der Corpus luteum Funktion spielen. Da eine solche Wirkung an die Expression spezifischer Rezeptoren gebunden ist, war es Ziel vorliegender Arbeit im Hinblick einer Verifizierung der aufgestellten These, die Expression von Progesteron- und Östrogenrezeptoren im Verlauf des Diöstrus im Corpus luteum ingravider Hündinnen zu erfassen. Die Untersuchungen erfolgten immunhistochemisch unter Verwendung spezifischer Antikörper und Einbeziehung externer und interner Kontrollen. Bei jeweils 4 Hündinnen unterschiedlicher Rassen wurden an den Tagen 5, 15, 25, 35 und 45 nach der Ovulation die Ovarien mittels Ovariohysterektomie entnommen, die Corpora lutea separiert, und für die Immunhistochemie in 4% Lilliformol fixiert und anschließend in Paraffin eingebettet. 3 µm dicke Schnitte, aufgezogen auf mit Aminopropyltriethoxysilan beschichtete Objektträger, wurden durch eine Xylol-Alkohol Reihe entparaffiniert, rehydriert und zur Demaskierung der Epitope einer Mikrowellenbehandlung mit Citratpuffer unterzogen. Zur Inaktivierung der endogenen Peroxidase folgte eine Überschichtung mit Wasserstoffperoxid in PBS-Puffer, zur Blockierung unspezifischer Proteinbindungsstellen die Überschichtung mit inaktivierten Pferdeserum; es schloss sich der immunhistochemische Rezeptornachweis an. Zum Nachweis von Progesteron- und Estradiol-17b-Rezeptoren kamen Antikörper aus dem Klon 10A9 bzw. 6F11 zur Anwendung. Externe Kontrolle war Uterusgewebe vom 15. Tag post ovulationem, interne Kontrollen waren die periluteal liegenden Follikel. Zur Überprüfung unspezifischer Bindung an caninem Gewebe wurden in jedem Test zusätzliche Schnitte mitgeführt, bei denen der spezifische Primärantikörper durch einen \u27nonsens\u27 Antikörper aus dem Klon 7T4-1F5 bzw. CBL-600 in entsprechender Endverdünnung des Proteins ersetzt wurde. Die lichtmikroskopische Auswertung der Präparate erfolgte bei 400-facher Vergrößerung. Pro Hund wurde ein Paraffinblock, d. h. ein Corpus luteum, herangezogen. Von diesem Block wurden 3 Schnitte gefärbt. Ausgezählt wurden je nach Schnitt 13 bis 21 Gesichtsfelder, wobei einem imaginären Kreuz gefolgt wurde. Die statistische Auswertung erfolgte mit dem Statistikprogrammpaket BMDP/Dynamic, Release 7.0 (Dixon 1993). Zu allen Untersuchungszeitpunkten ergaben sich Färbereaktionen, die das Vorkommen von PR und ER sowohl in den Luteinzellen als auch in den sonstigen Zellen anzeigten, wobei bei den Luteinzellen im Hinblick auf die nukleäre Reaktion zwischen \u27positiven\u27 und \u27schwach positiven\u27 Zellen differenziert werden konnte. Daneben ergaben sich auch zytoplasmatische Signale. Weiterhin zeigten sich, bezogen auf die Lokalisation, mehr positive Reaktionen in den Randfeldern als in den mittleren Feldern. Nur für den PR ließ sich ein Bezug zum Zeitpunkt der Probenentnahme feststellen, obwohl die Expression des ER positiv mit der des PR korreliert war. Die Progesteronkonzentration im peripheren Plasma korrelierte negativ mit der Zahl der PR exprimierenden Luteinzellen und sonstigen Zellen. Diese Befunde weisen erstmals darauf hin, dass beim Hund den lokal im CL gebildeten Sexualhormonen Progesteron und Estradiol-17b eine parakrine bzw. autokrine Funktion bei der Regulation der CL-Funktion zukommt. Inwieweit die Regulation der Expression der ER und PR im CL von den lokalen Verfügbarkeit von Progesteron und Estradiol-17b abhängt, kann derzeit nur spekulativ vermutet werden.In the dog neither an uterine luteolysine nor a lack of gonadotropins (LH, Prolaktin) seem to be functionally correlated to luteolysis which commences after the first third of diestrus. As a result of these observations the hypothesis was developed that in the dog, as in other species, steroid hormones of luteal origin act as local paracrine/autocrine factors controlling luteal function. In order to test for this hypothesis the present work deals with the determination of progesterone and estrogene receptors during the course of diestrus in corpora lutea of non-pregnant bitches. Immunhistochemical methods were applied using specific antibodies and adequate external and internal controls. Four bitches of different breeds were ovariohysterectomized on days 5, 15, 25, 35 and 45 after ovulation, corpora lutea were separated from the ovaries obtained, fixed with Lillie formole (4%) and embedded in paraffin; 3 µm slices were placed on slides coated with aminopropyltriethoxysilane. The samples were then deparaffinized and dehydrated by treatment with xylene and alcohol, followed by a microwave treatment in citrate-buffer for retrieval of epitopes. Endogenous peroxidase was inactivated by treatment with hydrogene peroxide in phosphate buffered solution, unspecific binding sites were blocked by incubation with horse serum prior to performing the immuncytochemical reaction. Antibodies derived from the clones 10A9 and 6F11 were used for detection of progesterone and estradiol-17b receptors, respectively. Uterine tissue obtained from a bitch 15 days after ovulation served as external control, follicles in periluteal position served as sample inherent (internal) controls. To test for unspecific bindig in additional control samples the specific primary antibody was replaced by a nonsens-antibody derived from the clones 7T4-1F5 and CBL-600, respectively. Nonsens and primary antibodies were used in the same dilution. All slides were evaluated using a 400-fold magnification. Evaluation was restricted to one corpus luteum per dog. From each corpus luteum 3 slides were prepared, evaluation followed an imaginary cross yealding between 13 to 21 views per slide. The program BMDP/Dynamic, Release 7.0 (Dixon 1993) was used for statistical evaluation. At all time-points investigated positive staining reactions were observed, indicating the presence of progesterone and estradiol receptors in luteal cells as well as in other cells. In respect to luteal cells a distinction was possible between positiv and weakly positiv nuclear reactions. Luteal cells also showed a cytoplasmic signal. There was a higher incidence of positive stained cells in the periphery and only the expression of the progesterone receptor was correlated to the stage of cycle. However, expression of the estrogene receptor was also positively correlated to the expression of the progesterone receptor. Progesterone concentrations in peripheral plasma were negatively correlated to the number of luteal cells expressing the progesterone receptor and the other progesterone receptor-positive cells. These are the first results indicating a paracrine/autocrine role of progesterone and estradiol in the regulation of luteal function in the dog. The observations warrant further studies to test for control of the expression of progesterone and estrogene receptors in the corpus luteum and the role of local luteal hormone concentrations

Fator de crescimento do endotélio vascular (VEGF): regulação transcricional e pós-transcricional.

O Fator de Crescimento do Endotélio Vascular (VEGF) desempenha importante papel regulador no desenvolvimento vascular fisiológico, sendo que tanto a diminuição nos seus níveis ou sua ausência quanto o aumento provocam danos na formação vascular sistêmica. É um fator angiogênico amplamente estudado, e em tecidos onde a angiogênese é aumentada sua expressão é consideravelmente alta. A expressão desse fator é regulada por processos transcricionais e pós-transcricionais incluindo o splicing alternativo, hipóxia, hormônios, outros fatores angiogênicos e microRNAs. Desse modo, esta revisão tem como objetivo relatar alguns dos principais mecanismos que controlam a produção do VEGF.Vascular Endothelial Growth Factor (VEGF) plays an important regulatory role in physiological vascular development, and either its levels reduction or its absence damages vascular formation. It is an widely studied angiogenic factor and in tissues where angiogenesis is increased, its expression is considerably high. The expression of this factor is regulated by transcriptional and posttranscriptional processes, including alternative splicing, hypoxia, hormones, other angiogenic factors and microRNAs. Thus the objective of this review is to report some of the most important mechanisms that control VEGF production

Growth factors and steroidogenesis in the bovine placenta

The control of placental hormone biosynthesis is critical during gestation, since their coordinated action is essential for the normal progress of pregnancy. Hormonal synthesis regulation in placenta is still not elucidated and differs from that observed in other steroidogenic tissues since specific tropic hormones have not yet been identified. Cellular localization of growth factors in the placenta, including VEGF, EGVEGF and bFGF, points that these factors have additional roles in the organ besides their well known modulation on cell proliferation and angiogenesis. In vitro experiments bring new evidence that growth factors play regulatory roles modulating processes related to steroid hormone secretion in the placenta. Importance of local estrogen function has been highlighted and a key enzyme for its synthesis is aromatase cytochrome P450. The objective of this review was to describe some aspects of placental steroidogenesis, mainly focusing on aromatase cytochrome P450 steroidogenic enzyme expression and growth factors as others potential modulators of hormonal synthesis in the orga

Description of an Ultrasound-Guided Erector Spinae Plane Block and Comparison to a Blind Proximal Paravertebral Nerve Block in Cows: A Cadaveric Study

The proximal paravertebral nerve block is commonly used to provide anaesthesia to the flank during standing surgical procedures in adult cattle. It has been reported that additional anaesthetic infiltration may be necessary to provide complete anaesthesia. In humans as well as animal species, another technique—the ultrasound (US)-guided erector spinae plane block (ESPB)—has been described. The goal of the present study was to develop and investigate an US-guided ESPB in comparison to a blind proximal paravertebral nerve block (PPNB) in cow cadavers. In 10 cadaver specimens, injections of methylene blue-lidocaine (1:1) were performed at the level of T13, L1 and L2 vertebras, on one side doing an ESPB block and, on the other side, a PPNB. Five cadavers were injected with high (40 mL per injection for PPNB and 20 mL for ESPB) and five with low (20 and 15 mL, respectively) volumes of injectate. For the ESPB, the ultrasound probe was oriented craniocaudally, and the ventral-cranial aspect of the articular processes (T13, L1 and L2) was targeted for injection. The dye spreading was evaluated by dissection. The landmarks for US-guided injection were easily visualized; however, injections were accidentally performed at T12, T13 and L1. Nevertheless, L2 was stained in 60% of ESPBs. Epidural spreading was observed with both techniques and all volumes. Viscera puncture was reported in two PPNBs. The ESPB resulted in similar nerve staining compared to the PPNB while using a lower volume of injectate. Even better staining is expected with a T13-L2 instead of a T12-L1 ESPB approach. Further studies are warranted to evaluate the clinical efficacy

Potential involvement of adinopectin and its receptors in the modulation of steroidogenesis in corpus luteum of bitches during diestrus

No ciclo estral de cadelas a fase luteínica, denominada diestro, compreende um período que varia de 60 a 100 dias em animais não-prenhes, caracterizado pela elevação plasmática de progesterona nos primeiros 20 dias pós ovulação (p.o). A adiponectina é a mais abundante proteína secretada pelo tecido adiposo, porém sua concentração plasmática diminui significativamente em alterações metabólicas como resistência insulínica e Diabetes mellitus tipo2, alterações descritas como relacionadas em algumas cadelas com o período de diestro. O objetivo do estudo foi determinar a expressão e imunolocalização do sistema adiponectina (adiponectina e seus receptores, adipoR1 e adipoR2) no corpo lúteo de cadelas ao longo do diestro, correlacionando-o ao perfil hormonal de 17β-estradiol e progesterona, assim como à expressão de um dos genes alvo do sistema, o PPAR-γ. Para realização do estudo foram coletados corpos lúteos de 28 cadelas durante ovariosalpingohisterectomia de eleição nos dias 10, 20, 30, 40, 50, 60 e 70 pós ovulação (o dia zero da ovulação foi considerado aquele no qual a concentração plasmática de progesterona atingiu 5ng/mL). Os corpos lúteos foram avaliados por imunohistoquímica para adiponectina e seus receptores e a expressão do RNAm do PPAR-γ por PCR em tempo real. A análise estatística da avaliação gênica foi realizada com o teste ANOVA, seguido por comparação múltipla Newman-Keuls. O sinal da adiponectina apresentou-se mais intenso até os primeiros 20 dias p.o, momento de regência da progesterona; houve queda gradativa após este período, coincidindo com a ascensão do 17β-estradiol, cujo pico foi notado próximo do dia 40 p.o. A queda marcante da adiponectina ocorreu após 50 dias p.o. O sinal do adipoR1 mostrou-se bem evidente até os 40 dias p.o e o do adipoR2 até os 50 dias p. o, decaindo posteriormente. Foi observada maior expressão do gene PPAR-γ aos 10, 30 e 70 dias p.o. Estes resultados mostram que a expressão protéica da adiponectina e de seus receptores se altera ao longo do diestro e que estas alterações podem estar relacionados às alterações hormonais e expressão do PPAR- γ, participando do mecanismo fisiológico de desenvolvimento, manutenção, atividade e regressão luteínica em cadelas.In the estrous cycle of bitches, the luteal phase or diestrus includes a period ranging from 60 to 100 days in non-pregnant animals, characterized by elevated serum progesterone during the first 20 days post-ovulation (p.o). Adiponectin is the most abundant protein secreted by adipose tissue, but plasma concentration decreases significantly in metabolic disorders like insulin resistance and diabetes mellitus type 2, described as related changes in some bitches in diestrus. The aim of this study was to determine the expression and immunolocalization of the adiponectin system (adiponectin, and adipoR1 adipoR2) in the corpus luteum during diestrus, and correlate it to hormonal profile of 17 beta-estradiol and progesterone, as well as the expression of a gene target of the system, the PPAR-gamma. For the study, corpora lutea were collected from 28 dogs during ovariosalpingohysterectomy on days 10, 20, 30, 40, 50, 60 and 70 post ovulation (day zero of ovulation was considered the day when the plasma progesterone concentration reached 5ng/mL). The corpora lutea were evaluated by immunohistochemistry for adiponectin, adipoR1 and adipoR2 and mRNA expression of PPAR-gamma by real-time PCR. Statistical analysis of gene expression was performed with ANOVA followed by Newman-Keuls multiple comparisons. Adiponectin positive signal was stronger during the first 20 days p.o, time of the regency of progesterone; there was a gradual adiponectin and progesterone decline after this period, coinciding with the rise of 17 beta-estradiol, whose peak was near the 40 days p.o. The markedly adiponectin decrease occurred after 50 days p.o. The signal of adipoR1 was markedly evident at 40 days p.o and that of adipoR2 up to 50 days p.o, declining afterwards. We observed higher expression of PPAR-gamma gene at 10, 30 and 70 days p.o. These results show that adiponectin and its receptors protein expression is altered during the diestrus and that these changes may be related to hormonal changes and expression of PPAR-gamma, participating in the physiological mechanism of development, maintenance, activity and luteal regression in bitches

An approach to uncover the relationship between 17b-estradiol and ESR1/ESR2 ratio in the regulation of canine corpus luteum

The canine corpus luteum (CL) is able to synthetise, activate and deactivate 17b-estradiol (E2) and also expresses nuclear estrogen receptors in a time-dependent manner during diestrus. Nevertheless, we are still missing a better comprehension of E2 functions in the canine CL, especially regarding the specific roles of estrogen receptor alpha (ERa) and ERb, encoded by ESR1 and 2, respectively. For that purpose, we analyzed transcriptomic data of canine non-pregnant CL collected on days 10, 20, 30, 40, 50 and 60 of diestrus and searched for differentially expressed genes (DEG) containing predicted transcription factor binding sites (TFBS) for ESR1 or ESR2. Based on biological functions of DEG presenting TFBS, expression of select transcripts and corresponding proteins was assessed. Additionally, luteal cells were collected across specific time points during diestrus and specificity of E2 responses was tested using ERa and/or ERb inhibitors. Bioinformatic analyses revealed 517 DEGs containing TFBS, from which 67 for both receptors. In general, abundance of predicted ESR1 targets was greater in the beginning, while abundance of ESR2 targets was greater in the end of diestrus. ESR1/ESR2 ratio shifted from an increasing to a decreasing pattern from day 30 to 40 post ovulation. Specific receptor inhibition suggested an ERa-mediated positive regulation of CL function at the beginning of diestrus and an ERb-mediated effect contributing to luteal regression. In conclusion, our data points toward a broad spectrum of action of E2 and its nuclear receptors, which can also act as transcription factors for other genes regulating canine CL function

Morfologia dos órgãos genitais do macho do Lobo marinho (Arctocephalus australis) e implicações biológicas

Male capacity for spreading genes to a great number of descendents and to determine population dynamics depend directly on the genital organs. Morphological studies in pinnipeds are scarce and the functional meaning of some characteristics has never been discussed. We hypothesized that Arctocephalus australis (A. australis) shows morphophysiological adaptations in order to guarantee the perpetuation of the species in the unique annual mating season. Seven males, dead from natural causes, had their genital organs collected and fixed for morphological description. Some features differ from other described mammalian males and are closely related to the biology and reproductive cycle of this species, as the scrotal epidermis, absence of glandular portion in the ductus deferens and spermatogenic epithelium suggest a recrudescent testis period. The corona glandis exhibits a singular arrangement: its erectile border looks like a formation of petals and its association with the os penis gives a "lily-flower" form to this region. We propose the name margo petaliformis to this particular erectile border of the corona glandis because of its similarity to a flower corola. The male genital organs of A. australis show morphological features compatible with adaptation to environment requirements and reproductive efficiency

Aspectos biométricos do desenvolvimento testicular e corporal em cutias (Dasyprocta aguti) criadas em cativeiros

Biometric data of the testicular and corporal weight development of 31 Agoutis (Dasyprocta aguti), from the birth up to 14 months of age, were analyzed. The correlations between corporal weight, age and testicular parameters were highly significant. Testicular weight, testicular volume, as well other testicular biometric parameters (length, diameter and perimeter), evolved slow and gradually until eight months of age. Beginning on 9 months of age, the growth was faster, and in this period. the seminiferous epithelium was already formed. Biometric development of the testes can be shared in two phases: 0 - 8 months and 9 - 14 months of age, however the 9 months age are the maximum point of the testicular development in Agoutis (Dasyprocta aguti) rised in captivity.Analisou-se os dados biométricos do desenvolvimento testicular e peso corporal de 31 cutias (Dasyprocta aguti) desde o nascimento até os 14 meses de idade. As correlações entre o peso corporal, idade e parâmetros testiculares apresentaram-se altamente significativas. O peso testicular, o volume testicular, assim como os demais parâmetros biométricos testiculares (comprimento, diâmetro e perímetro), evoluíram lenta e gradualmente até os 8 meses de idade. A partir dos 9 meses, o crescimento foi mais rápido. O desenvolvimento biométrico do testículo pode ser dividido em duas fases, de 0 - 8 meses e de 9 - 14 meses de idade, sendo 9 meses considerado ponto de corte em se tratando de desenvolvimento testicular de cutias criadas em cativeiro

Análise qualitativa do estabelecimento da espermatogênese em cutias (Dasyprocta aguti) criadas em cativeiros

A determinação do estabelecimento da puberdade é bastante estudada em animais domésticos e roedores, no entanto, são escassas as pesquisas com a finalidade de estabelecer parâmetros para a biologia reprodutiva em cutias. Foram utilizadas 31 cutias machos da espécie Dasyprocta agouti, oriundas da Universidade Federal do Piauí, Estado do Piauí, e da Escola Superior de Agricultura de Mossoró, Estado do Rio Grande do Norte. Imediatamente após a orquiectomia foram retirados fragmentos e estes foram processados histologicamente, os tecidos foram corados com hematoxilina-eosina e analisou-se os parâmetros seguintes: aspectos de luminação dos túbulos seminíferos; presença de espermatócitos primários; presença de espermátides e formação dos primeiros estágios do ciclo do epitélio seminífero (CES) segundo o método da morfologia tubular. O período desde o nascimento até os cinco meses de idade correspondeu à fase impúbere; dos seis aos oito meses de idade a fase de transição da pré-puberdade à puberdade; dos nove aos dez meses de idade à fase da puberdade; e dos doze aos quartoze meses de idade à fase da pós-puberdade. A puberdade da cutia (Dasyprocta aguti), ocorreu em animais a partir dos sete meses de idade, e o estabelecimento da puberdade foi constatado em todos os animais estudados aos nove meses de idade.The determination of the puberty beginning is widely studied in domestic animals and rodents. However, there are scarce researches with the purpose to establish parameters for the reproductive biology in Agoutis. Thirty-one male Agoutis, Dasyprocta agouti species had been used, originated from the Universidade Federal do Piauí - PI, and from the Mossoró Superior Scholl of Agriculture - RN. Immediately after the orchiectomy, histological sections were made, the tissues were stained with hematoxylin-eosine and analyzed for the following parameters: aspects of the seminiferous tubule lumen; presence of primary spermatocytes; presence of spermatid cells and formation of the first stages of the seminiferous epithelium cycle (SEC), in accordance with the tubular morphology method. The period from the birth up to five months of age corresponded to the impuberal phase; from six to eight months, to the transition from the prepuberal phase to puberty; from nine to ten months, to the puberty; and, from twelve to fourteen months of age to the after-puberty phase. The puberty of the Agouti (Dasyprocta aguti), occurred since seven months of age, being the establishment puberty beginning evidenced at nine months of age

Quantificação de células dos túbulos seminíferos e rendimento da espermatogênese em cutias (Dasyprocta aguti) criadas em cativeiros

This study has as objective to evaluate the spermatogenesis yield of Agoutis rised in captivity, through the rates found between cellular types of the seminiferous epithelium. The results showed that the spermatogenesis yield of the Agoutis since 9 to 14 months of age did not reach the stabilization point. The coefficient of efficiency of the spermatogonium mitoses, did not increase with the age. The meiotic yield, usual spermatogenesis yield and the Sertoli cells index didn't showed numeric variation at function of the age, however, it was not detected by statistic data.O presente estudo teve como objetivo avaliar o rendimento da espermatogênese de cutias criadas em cativeiro, por intermédio das razões encontradas entre tipos celulares do epitélio seminífero. Os resultados apontaram que o rendimento da espermatogênese da cutia dos nove aos quatorze meses de idade não chegou a um ponto de estabilização. O coeficiente de eficiência de mitoses espermatogoniais não aumentou com a idade. O rendimento meiótico, o rendimento geral da espermatogênese e o índice de células de Sertoli mostraram variações numéricas em função da idade, entretanto, não detectadas estatisticamente