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.

Equine Chorionic Gonadotropin Modulates the Expression of Genes Related to the Structure and Function of the Bovine Corpus Luteum.

We hypothesized that stimulatory and superovulatory treatments, using equine chorionic gonadotropin (eCG), modulate the expression of genes related to insulin, cellular modelling and angiogenesis signaling pathways in the bovine corpus luteum (CL). Therefore, we investigated: 1-the effect of these treatments on circulating insulin and somatomedin C concentrations and on gene and protein expression of INSR, IGF1 and IGFR1, as well as other insulin signaling molecules; 2-the effects of eCG on gene and protein expression of INSR, IGF1, GLUT4 and NFKB1A in bovine luteal cells; and 3-the effect of stimulatory and superovulatory treatments on gene and protein expression of ANG, ANGPT1, NOS2, ADM, PRSS2, MMP9 and PLAU. Serum insulin did not differ among groups (P = 0.96). However, serum somatomedin C levels were higher in both stimulated and superovulated groups compared to the control (P = 0.01). In stimulated cows, lower expression of INSR mRNA and higher expression of NFKB1A mRNA and IGF1 protein were observed. In superovulated cows, lower INSR mRNA expression, but higher INSR protein expression and higher IGF1, IGFR1 and NFKB1A gene and protein expression were observed. Expression of angiogenesis and cellular modelling pathway-related factors were as follows: ANGPT1 and PLAU protein expression were higher and MMP9 gene and protein expression were lower in stimulated animals. In superovulated cows, ANGPT1 mRNA expression was higher and ANG mRNA expression was lower. PRSS2 gene and protein expression were lower in both stimulated and superovulated animals related to the control. In vitro, eCG stimulated luteal cells P4 production as well as INSR and GLUT4 protein expression. In summary, our results suggest that superovulatory treatment induced ovarian proliferative changes accompanied by increased expression of genes providing the CL more energy substrate, whereas stimulatory treatment increased lipogenic activity, angiogenesis and plasticity of the extracellular matrix (ECM).

Is the canine corpus luteum an insulin-sensitive tissue?

This study aimed to determine in the canine corpus luteum throughout the dioestrus (1) the influence of insulin on glucose uptake; (2) the regulation of genes potentially involved; and (3) the influence of hypoxia on glucose transporter expression and steroidogenesis, after treatment with cobalt chloride (CoCl2). Glucose uptake by luteal cells increased 2.7 folds (P < 0.05) in response to insulin; a phenomenon related to increased expression of glucose transporter (GLUT) 4 and phosphorylation of protein kinase B (AKT). The gene expression of insulin receptor and SLC2A4 (codifier of GLUT4) genes after insulin stimulation increased on day 20 post ovulation (p.o.) and declined on day 40 p.o. (P < 0.05). Regarding potentially involved molecular mechanisms, the nuclear factor kappa B gene RELA was upregulated on days 30/40 p.o., when SLC2A4 mRNA was low, and the interleukin 6 (IL6) gene was upregulated in the first half of dioestrus, when SLC2A4 mRNA was high. CoCl2 in luteal cell cultures increased the hypoxia-inducible factor HIF1A/HIF1A and the SLC2A4/GLUT4 expression, and decreased progesterone (P4) production and hydroxyl-delta-5-steroid dehydrogenase 3 beta (HSD3B) mRNA expression (P < 0.05). This study shows that the canine luteal cells are responsive to insulin, which stimulates glucose uptake in AKT/GLUT4-mediated pathway; that may be related to local activity of RELA and IL6. Besides, the study reveals that luteal cells under hypoxia activate HIF1A-modulating luteal function and insulin-stimulated glucose uptake. These data indicate that insulin regulates luteal cells' glucose disposal, participating in the maintenance and functionality of the corpus luteum.

Microvascularization of corpus luteum of bovine treated with equine chorionic gonadotropin.

This study aimed to evaluate the morphological changes in microvascular density and corpus luteum (CL) vascularization in cows treated with eCG during stimulatory and superovulatory protocols. Sixteen cows were synchronized and divided into three groups: control (n = 6), stimulated (n = 4) and superovulated (n =6), one was submitted to estrous synchronization (ES) and received no eCG (control), and those that were submitted to ES and received eCG before or after follicular deviation (superovulation and stimulation of the dominant follicle, respectively). Ovulation was synchronized using a progesterone device-based protocol. After six days of ovulation, the cows were slaughtered and the ovaries and CL were collected. The CLs were processed and photomicrographs were taken under light microscopy to assess the vascular volume density (Vv) by stereology, and scanning electron microscopy (SEM) was used to perform ultrastructural analysis of the microvasculature. The Vv in stimulated and superovulated cows significantly increased (P ≤ 0.0001) when compared to control, indicating that the eCG is able to induce angiogenic activity in bovine CL. However, no significant differences were observed between stimulated and superovulated cows. The SEM demonstrated ratings indicative of angiogenesis, marked by several button-shaped projections in the capillaries, and the presence of more dilated capillaries in CL treated with eCG. These morphological findings are evidence of an angiogenic effect of the eCG treatment in CL of cows.

Regulation of the polyamine metabolic pathway in the endometrium of cows during early diestrus.

The timing and magnitude of exposure to preovulatory estradiol followed by post-ovulatory progesterone (periovulatory endocrine milieu) in cattle modulate endometrial gene expression, histotroph composition, and conceptus development, but the mechanisms underlying this regulation remain unknown. Using an experimental model based on the modulation of follicle growth, this work aimed to evaluate if the polyamine metabolic pathway is regulated by the periovulatory endocrine milieu. Nelore cows were manipulated to ovulate small (n = 15) or large (n = 15) follicles, then the profiles of polyamines and their synthetic enzymes were compared between groups. Transcripts for the enzymes of this pathway, ornithine decarboxylase 1 (ODC1; the rate-limiting enzyme in polyamine biosynthesis) protein quantification, adenosylmethionine decarboxylase 1 (AMD1) protein immunolocalization, and concentrations of the different polyamines (putrescine, spermidine, and spermine) were respectively quantified by quantitative reverse-transcriptase PCR, immunoblotting, immunohistochemistry, and gas chromatography-mass spectrometry in both the endometrium and uterine flushing. No differences in gene and protein expression or concentration of polyamines were observed between groups. There were significant correlations between the relative abundance of ODC1 and spermidine/spermine N1-acetyltransferase 1 (SAT1) transcripts as well as between antizyme inhibitor 1 (AZIN1) and adenosylmethionine decarboxylase 1 (AMD1) transcripts. In conclusion, our results show that the polyamine metabolic pathway is present and functional, but not regulated by the periovulatory endocrine milieu in the bovine endometrium.

Glucose transporter 1 expression accompanies hypoxia sensing in the cyclic canine corpus luteum.

The canine corpus luteum (CL) functions as a source of progesterone (P4) and 17ß-oestradiol (E2); however, the transport of energy substrates to maintain its high hormonal output has not yet been characterised. This study involved the localisation and temporal distribution of the facilitative glucose transporter 1 and the quantification of the corresponding protein (GLUT1) and gene (SLC2A1) expression. Some GLUT1/SLC2A1 regulatory proteins, such as hypoxia-inducible factor 1α (HIF1A) and fibroblast growth factor 2 (FGF2); mRNAs, such as HIF1A, FGF2 and vascular endothelial growth factor A (VEGFA); and VEGFA receptors 1 and 2 (FLT1 and KDR) were also analysed from days 10 to 70 after ovulation. Additionally, plasma P4 and E2 levels were assessed via chemiluminescence. Moreover, the canine KDR sequence has been cloned, thereby enabling subsequent semi-quantitative PCR analysis. Our results demonstrate time-dependent variations in the expression profile of SLC2A1 during dioestrus, which were accompanied by highly correlated changes (0.84

Genital morphology of the male South American fur seal (Arctocephalus australis) and biological implications / 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.(AU)

A capacidade do macho de espalhar seus genes a um grande número de descendentes e determinar a dinâmica populacional depende diretamente dos seus órgãos genitais. Estudos morfológicos em pinípedes são escassos e o significado funcional de algumas de suas características ecológicas ainda foi pouco discutido. Nossa hipótese é que Arctocephalus australis (A. australis) apresenta adaptações morfofisiológicas em seus órgãos genitais capazes de interagir com o meio e garantir a perpetuação da espécie que apresenta apenas uma época de acasalamento que ocorre uma vez a cada ano. Sete A. australis machos, mortos recentes por causas naturais, tiveram seus órgãos genitais coletados e fixados para a descrição macro, micro e ultraestrutural. Algumas características diferem de outros machos já descritos e estão intimamente relacionados com a biologia e ciclo reprodutivo da espécie, dentre elas podemos citar a alta queratinização da epiderme escrotal que pode se relacionar com as rotineiras lesões por atrito desta região nas pedras; a ausência da porção glandular do ducto deferente aqui descrita pela primeira vez, o epitélio espermatogênico sugere um período de testículo recrudescente. A glande apresenta um arranjo singular: a coroa da glande apresenta porção lateral de tecido esponjoso que são bordas livres com capacidade de intumescencia. O osso peniano se encontra no centro destas bordas e representa a extremidade mais distal do penis, levando consigo o óstio uretral externo. As bordas associadas ao osso peniano, dão uma forma de "Flor de lírio" a esta região. Utilizamos o nome margo petaliformis a margem erétil liliforme a particular morfologia da glande, pela sua semelhança a uma corola de flor. Os órgãos genitais masculinos de A. australis mostram características morfológicas compatíveis com uma adaptação aos requisitos ambientais e de eficiência reprodutiva.(AU)

Morphological features and vascularization study of caprine cyclic corpus luteum / Características morfológicas e estudo da vascularização do corpo lúteo cíclico de cabras ao longo do ciclo estral

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 percent 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.

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 por cento 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.

Aspectos biométricos do desenvolvimento testicular e corporal em cutias (Dasyprocta aguti) criadas em cativeiros / Biometric aspects of the testicular and corporal development of 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.

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.

Quantificação de células dos túbulos seminíferos e rendimento da espermatogênese em cutias (Dasyprocta aguti) criadas em cativeiros / Histologic quantification of the seminiferous tubules cells and spermatogenesis yield in Agoutis (Dasyprocta aguti) rised in captivity

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.

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.

Análise qualitativa do estabelecimento da espermatogênese em cutias (Dasyprocta aguti) criadas em cativeiros / Qualitative analisis of the established spermatogenesis in agoutis (Dasyprocta aguti) rised in captivity

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.