Morphological features and vascularization study of caprine cyclic corpus luteum

Pesquisa Veterinária Brasileira

Freitas, Maria de Lourdes¹  Rocha, Hugo Alexandre de Oliveira²  Miranda-Moura, Maria Tereza M.¹  Almeida, Osiel Benedito de¹  Fonseca, Vanessa Uemura da³  Silva, Naisandra Bezerra da¹  Moura, Carlos Eduardo Bezerra de¹  Papa, Paula de Carvalho³ 

[1] Universidade Federal do Rio Grande do Norte, Natal, Brazil;UFRN, Natal;Universidade de São Paulo, São Paulo, Brazil

关键词: Corpus luteum;    goat;    anatomy;    vascular density;    progesterone.;   

DOI  :  10.1590/S0100-736X2010000400012

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

Corpus luteum is a temporary endocrine gland that regulates either the estrous cycle and pregnancy. It presents extreme dependency on the adequate blood supply. This work aims to evaluate goat corpus luteum (CL) vascular density (VD) over the estrous cycle. For that purpose, 20 females were submitted to estrus synchronization/ovulation treatment using a medroxyprogesterone intra-vaginal sponge as well as intramuscular (IM) application of cloprostenol and equine chorionic gonadotrophine (eCG). After sponge removal, estrus was identified at about 72hs. Once treatment was over, female goats were then subdivided into 4 groups (n=5 each) and slaughtered on days 2, 12, 16 and 22 after ovulation (p.o). Ovaries were collected, withdrawn and weighted. CL and ovaries had size and area recorded. Blood samples were collected and the plasma progesterone (P4) was measured through RIA commercial kits. The VD was 24.42±6.66, 36.26±5.61, 8.59±2.2 and 3.97±1.12 vessels/mm2 for days 2, 12, 16 and 22 p.o, respectively. Progesterone plasma concentrations were 0.49±0.08, 2.63±0.66, 0.61±0.14 and 0.22±0.04ng/ml for days 2, 12, 16 e 22 p.o, respectively. Studied parameters were affected by the estrous cycle phase. Values greater than 12 p.o were observed. In the present work we observed that ovulation occurred predominantly in the right ovary (70% of the animals), which in turn presented bigger measures than the contra lateral one. There is a meaningful relationship between the weight and size of the ovary and these of CL (r=0.87, r=0.70, respectively, p<0.05). It is possible to conclude that morphology of goat's ovaries and plasma progesterone concentration changed according to estrous cycle stages. We propose these parameters can be used as indicators of CL functional activity.Index terms: Corpus luteum, goat, anatomy, vascular density, progesterone. RESUMO O corpo lúteo é uma glândula endócrina temporária que regula tanto o ciclo estral quanto a prenhez, apresentando extrema dependência de aporte sanguíneo adequado. Objetivaram-se avaliar mudanças morfométricas dos ovários e densidade vascular (DV) dos corpos lúteos (CL) de cabras ao longo do ciclo estral (AOLC). Vinte animais foram submetidos ao tratamento para indução/sincronização do estro, usando esponjas intravaginais commedroxiprogesterona, associadas a aplicações intramusculares de cloprostenol e gonadotrofina coriônica eqüina. Após remoção das esponjas, o estro foi identificado em aproximadamente de 72h. Concluído o tratamento, as cabras foram subdivididas em 4 grupos (n=5 cada) para abate nos dias 2, 12, 16 e 22 após ovulação (p.o.). Posteriormente, foram retirados os ovários e realizadas as mensurações de peso, tamanho e área do órgão e dos CL. Amostras de sangue foram coletadas e a progesterona sérica (P4) mensurada utilizando-se RIA convencional. A DV média dos CL AOLC foi 24,42±6,66; 36,26±5,61; 8,59±2,2 e 3,97±1,12 vasos/mm2 para os dias 2, 12, 16 e 22 p.o., respectivamente. A concentração média de P4 foi de 0,49±0,08; 2,63±0,66; 0,61±0,14 e 0,22±0,04ng/ml para os dias 2, 12, 16 e 22 p.o., respectivamente. Os parâmetros em estudo também se mostraram afetados pela fase do ciclo estral, sendo observados os maiores (p < 0,05) valores no dia 12 p.o. Neste experimento, a ovulação ocorreu predominantemente no ovário direito (70% dos animais), o qual apresentou medidas maiores que o contralateral. Observou-se ainda alta correlação significativa entre o peso do ovário e o do CL (r=0,87; p<0,05) e entre o tamanho destes órgãos (r=0,70; p<0,05). Conclui-se que, a morfologia dos ovários de cabras e a concentração sérica de progesterona variam em função da fase do ciclo estral e podem ser utilizadas como parâmetro na avaliação funcional do órgão.Termos de indexação: Corpo lúteo, caprino, anatomia, densidade vascular, progesterona.     INTRODUCTIONIn the last years, biotechnological research has focused on the development of techniques and procedures enabling increase of reproductive efficiency in economical important animals. Morphological studies describing the organization of the female reproductive system as well as histological and physiological changes related to the estrous cycle are the basis of reproductive oriented research.Among economical important species, the goat has gained a lot of attention since the start of putative increase, according to Food and Agriculture Organization of the United Nations (FAO 2000), goat population is estimated in 700 millions, from which 92% are distributed in tropical and subtropical underdeveloped areas. Even though goat herds are spread all over the world, they usually are treated empirically and with low levels of technology.In order to assure the success of goats' biotechnical exploitation, it is necessary to achieve reproductive efficiency, which can be measured by an increase in the pregnancy number during the female reproductive life. Thus, it is fundamental to understand the estrous cycle characteristics of the species, especially in events related to the development, life span and regression of the corpus luteum (CL).The corpus luteum is one endocrine organ showing a limited, short existence. It plays a central role in the regulation of estrous cycle and pregnancy by secreting progesterone, which should be maintained in appropriate levels in mammals in order to achieve a successful reproduction (Stocco et al. 2007).Angiogenesis is an essential event for CL formation and maintenance as well as for progesterone production (Yan et al. 1998). Besides, it is known that there is an intimate correlation between CL vascularization, blood flow and hormone production (Stormshak et al. 1963, Kobayashi et al. 2001). CL angiogenesis is controlled by a number of growth factors including VEGF (vascular endothelial), FGF (fibroblasts grown factor), TGF-ß (transforming growth factor), FGF (fibroblast growth factor), PDGF (platelet derived growth factor), interleukin-8 and angiopoietin-1 (Stocco et al. 2007).The morphometrical characterization of the reproductive organs is important to highlight variations occurring in ovarian structures responsible for gametogenesis and esteroidogenesis during estrous cycle and pregnancy. During the estrous cycle there are growth and atresia of a variable follicle number (Ginther et al. 1989, Roche et al. 1991), formation and regression of corpus luteum (Kastelic et al. 1990), phenomena which influence directly morphological characteristics of the ovary. The morphological characteristics of the ovary can also be useful to verify the pathological presence of cists and tumors as well as to determine the presence of a cyclic ovarian activity and even to estimate the probable cycle phase, allowing one to infer on the animal's fertility status (Nascimento et al. 2003).The hypothesis was that goat ovary and CL morphological changes reflect reproductive functional status. To test this hypothesis, this work aimed to describe ovarian macroscopic morphological parameters, to determine CL vascular density and plasma progesterone concentrations (as a marker of functional status) in this species.  MATERIALS AND METHODSAnimals and synchronization treatment/estrous induction.During the months of March and April 2006, 20 cross breed adult goats presenting a recorded healthy reproductive history, were confined in the Pedra Vermelha Farm, Lages, Rio Grande do Norte, Brazil. Animals were submitted to estrous induction/ synchronization treatment as described by Freitas & Lopes Jr (2002). Briefly, at day zero (d 0) of treatment, animals received intravaginally 60mg of medroxiprogesterona acetate sponges (MAP, Progespon® Tecnopec, São Paulo). After seven days (d 7), each animal received 300 UI of equine chorionic gonadotrophine (eCG, Novormon® Tecnopec, São Paulo, Brazil) as well as 5ml of cloprostenol (Prolise® Tecnopec, São Paulo) intramuscularly. After 48 h (d 9), sponges were removed and the estrus was observed within 72 h and considered the day of ovulation.Ovarian morphometrical samples and corpus luteum collection. The goats were divided into four groups (n=5 in each group) and slaughtered on days 2, 12, 16 and 22 post ovulation (p.o.). The ovaries were colletected immediately after evisceration, dissected and measured using a paquimeter precision scale. The symmetry of the organs was respected. Luteal tissue mass was calculated using the ellipse area defined by the highest and lowest diameter of the CL (Nascimento et al. 2003). Blood samples were collected by puncture of the jugular vein and centrifuged at 4000rpm, for 5min under 5ºC and kept at 20º until plasma progesterone was assessed by RIA.CL microscopic examination. CL was dissected and cut into pieces of 5cm3, which were fixed in 4% phosphate buffered formalin solution, pH 7.2 for 24 hours. Paraffin embedding was preceded by dehydration in increasing ethanol concentrations as well as xylene clarification.Slices with 6µm from paraffin blocks were obtained and stained with hematoxiline-eosine (HE) and Schiff's periodical acid (PAS) reaction.Vascular density determination. Vascular density (expressed in number of vessels per area in cm2) was determined as proposed by Ferreira-Dias et al. (2006). Six µm slices were stained by PAS, which strongly reacts with basal membrane carbohydrates and is widely used as a marker for endothelial cells (Luna et al. 1992).For each animal, 16 fields (0.77cm2 area) were counted. These were chosen randomly through 6 histological sections, using a 400x magnification and an image reader software (Imagen-pro Plus®). All vessels in luteal tissue were equally evaluated (arterioles, venules e capillaries).Plasma progesterone concentration. Progesterone concentration in plasma sample was evaluated using a commercial kit (DSL-3400â, Diagnostic Systems Laboratories, USA). The standard curve was obtained by P4 increasing concentrations diluted in a phosphate gel buffer (PBS-G). The radioactive [I-125] progesterone was diluted at 70% in PBS-G and the primary antibody (rabbit origin) and the precipitation liquid (goat origin) were deluded at 60% in PBS-G. As positive control, diluted follicular fluid raging from low to medium and high progesterone content was used.The results were calculated using the Riasmart program. The intra and inter-assay variation coefficient were 6.14% and 5.34%, respectively.Statistical analysis. All the results were expressed as average ± standard deviation. Statistical comparison was done using ANOVA. The multiple comparison Tu

【 授权许可】

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