Factors affecting the fate of the canine corpus luteum: Potential contributors to pregnancy and non-pregnancy.

The fate of the canine corpus luteum (CL) differs from that of other domestic species: beyond the extended luteal regression observed in both pregnant and non-pregnant cycles, active luteolysis is observed only in pregnant dogs. Luteal regression in the absence of pregnancy lacks a luteolytic trigger. The CL lifespan during pregnancy is around 60 days, as long as that of the cyclic CL. Although they are already available in the first half of diestrus, LH and especially prolactin (PRL) play a decisive luteotropic role from approximately day 25 post-ovulation onwards. Nevertheless, many locally-produced factors are orchestrated to ensure a fully functional CL, which in the bitch produces progesterone (P4), 17b-estradiol, and other local regulators. Recently, insulin has been described as another luteotropic factor in this species, able to increase glucose uptake in luteal cells and contribute to steroid biosynthesis. The locally-produced PGE2 is also a potent luteotropic factor in the first half of diestrus, promoting STAR expression, as are also proliferating, vasoactive- and immunomodulatory factors. These, in turn, all contribute to the formation and maintenance of the canine CL. Meanwhile PGF2a, produced by the utero-placental compartment, participates actively in triggering pre-partum luteolysis. Cytokines play different roles, either contributing as luteotropic or as acute inflammation molecules. So far, the one clinically most efficient mechanism of interrupting a pregnancy in the dog is to block P4 receptors, using an antigestagen (e.g., aglepristone) in the second half of diestrus. To enhance the chances of pregnancy, however, several luteotropic factors could be used.

Tannic Acid Solution: A Better Fixative Solution Than Formalin for Elastin and Collagen-Toxic and Morphological Assessment.

Formaldehyde is commonly used worldwide, even though it is classified as carcinogenic to humans by the International Agency for Research on Cancer. This has motivated intensive investigations of formaldehyde substitutes, and recently, some alternative solutions were found, which can potentially replace it. Previous research showed that tannic acid (TA) in glutaraldehyde solution has the ability to stabilize elastin and collagen. This provided a basis for the development of a new alcoholic fixative solution, particularly aimed at extracellular matrix components, with TA as a main component. Heart, brain, and intestinal samples were fixed by immersion in 10% regular formalin solution (RFS), 70% ethanol solution (ES), and tannic acid ethanolic solution (TAES). Next, tissue fragments were prepared for routine histology procedures. The toxicity of TA was analyzed using in silico tests for mutagenicity, as well as for cutaneous and respiratory toxicity. Analyses of photomicrographs demonstrated that all fixative solutions have the ability to preserve the fragments. The quantitative analyses showed that capability of TAES to preserve and stabilize elastin and collagen is superior to that of RFS and ES. We demonstrated that TA is not mutagenic, and it is less toxic for skin and respiratory tract. We therefore conclude that TAES can potentially represent a powerful and feasible alternative solution for fixing extracellular matrix of microscopic examination samples. Anat Rec, 301:1544-1550, 2018. © 2018 Wiley Periodicals, Inc.

Evaluation of cell proliferation and endometrial thickness of bitches in different periods of diestrus

ABSTRACT This study aimed to determine the histological features of the endometrium of bitches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). In the present study, it is concluded that endometrial thickness does not differ at any of the moments analyzed in diestrus. The endometrial thickness is not influenced by hormones, weight, blood glucose or serum cholesterol of bitches in this phase of the estrous cycle. However, there is greater cell proliferation in the endometrium at day 40 compared to day 60 post ovulation under the influence of the endocrine profile.

Evaluation of cell proliferation and endometrial thickness of bitches in different periods of diestrus.

This study aimed to determine the histological features of the endometrium of bitches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). In the present study, it is concluded that endometrial thickness does not differ at any of the moments analyzed in diestrus. The endometrial thickness is not influenced by hormones, weight, blood glucose or serum cholesterol of bitches in this phase of the estrous cycle. However, there is greater cell proliferation in the endometrium at day 40 compared to day 60 post ovulation under the influence of the endocrine profile.

Morphometric and high resolution scanning electron microscopy analysis of low-level laser therapy and latex protein (Hevea brasiliensis) administration following a crush injury of the sciatic nerve in rats.

This study evaluated the effect of low-level laser therapy (LLLT; 15 J/cm(2)) and a latex protein (F1) on a crush injury of the sciatic (ischiadicus) nerve. Seventy-two rats (male, 250 g) were divided into 6 groups: CG, control; EG, exposed nerve; IG, injured nerve without treatment; LG, injured nerve with LLLT; HG, injured nerve with F1; and LHG, injured nerve with LLLT and F1. After 4 or 8 weeks, the animals were euthanized and samples of the sciatic nerve were collected for morphometric and high-resolution scanning electron microscopy (HRSEM) analysis. After 4 weeks, the morphometry revealed improvements in the treated animals, and the HG appeared to be the most similar to the CG; after 8 weeks, the injured groups showed improvements compared to the previous period, and the results of the treatment groups were more similar to one another. At HRSEM after 4 weeks, the treated groups were similar and showed improvement compared to the IG; after 8 weeks, the LHG and HG had the best results. In conclusion, the treatments resulted in improvement after the nerve injury, and this recovery was time-dependent. In addition, the use of the F1 resulted in the best morphometric and ultrastructural findings.

Relationship between adiponectin and fertility in the female pig.

Adiponectin isoforms may mediate different aspects of the pleiotropic function of the protein, including the reproductive process. We examined the pattern of circulating adiponectin and adiponectin system expression in fat and ovarian tissues of hyperfertile and subfertile sows. We demonstrated the presence of five different isoforms of adiponectin (90, 158, 180, 250 and >250kDa) in the circulation and identified a subgroup of subfertile females that displayed reduced abundance of all adiponectin isoforms as well as a lack of the 250-kDa adiponectin isoform in both serum and follicular fluid. Subfertility in these animals was associated with fewer large follicles and corpora lutea in the ovaries, as well as lower concentrations of 17ß-oestradiol in the follicular fluid of large follicles. In addition, subfertile females showed higher adiponectin mRNA in fat tissue and altered mRNA and protein expression of adiponectin and its receptors in the ovary. Changes in the abundance and pattern of circulating adiponectin isoforms have been associated with reproductive disorders in animals and humans, including polycystic ovarian syndrome (PCOS). Our findings suggest that the adiponectin system may play an important role in controlling ovarian function and influencing porcine fertility.

VEGF system expression by immunohistochemistry and real-time RT-PCR study on collared peccary placenta.

Vascular endothelial growth factor (VEGF) is known to induce endothelial cell proliferation, to promote cell migration, and to inhibit apoptosis, thus playing a central role in angiogenesis and in the regulation of vasculogenesis. The expression of the VEGF-ligand receptor system was studied in the placenta and uterus of the collared peccary in nonpregnant females in the luteal phase and throughout pregnancy (>35, 75, 115, and 135 days). The material was examined by immunohistochemistry and by real-time reverse transcription polymerase chain reaction. Intense positive immunolabeling was observed for VEGF and its receptors in the uterine epithelium, uterine glands, and trophoblast. The endothelial cells and smooth muscle cells in the maternal and fetal vessels, as well as the connective tissue and mesenchyme, had weak immunoreactivity during all periods of pregnancy. The regression analysis of the real-time polymerase chain reaction results demonstrated cubic behavior, showing a specific time-dependent profile during pregnancy, which increased over the last gestational period to VEGF and VEGFR-1. The relative expression of VEGFR-2 decreased in the middle-third of the pregnancy and increased in late pregnancy. In the collared peccary, the expression of the VEGF-ligand receptor system was similar to that in porcine and ruminant placentas, suggesting that an epitheliochorial placenta has the same physiological and interhemal barrier during vascular gestational development. The expression of VEGF among cells not related to the vascular system, such as those of the uterine epithelium, trophoblast, and uterine glands, suggests a distinct regulatory role for these cells in vasculogenesis and also a different role of VEGF pathway.

Equine chorionic gonadotropin alters luteal cell morphologic features related to progesterone synthesis.

Exogenous eCG for stimulation of a single dominant follicle or for superovulation are common strategies to improve reproductive efficiency by increasing pregnancy rates and embryo production, respectively. Morphofunctional changes in the CL of eCG-treated cattle include increases in CL volume and plasma progesterone concentrations. Therefore, we tested the hypothesis that eCG alters the content of luteal cells and mitochondria related to hormone production. Twelve crossbred beef cows were synchronized and then allocated into three groups (four cows per group) and received no further treatment (control) or were given eCG either before or after follicular deviation (superovulation and stimulation of the dominant follicle, respectively). Six days after ovulation, cows were slaughtered and CL collected for morphohistologic and ultrastructural analysis. Mitochondrial volume per CL was highest in superovulated followed by stimulated and then control cows (18,500 ± 2630, 12,300 ± 2640, and 7670 ± 3400 µm(3); P < 0.001), and the density of spherical mitochondria and the total number of large luteal cells were increased (P < 0.05) in stimulated cows compared with the other two groups (110.32 ± 14.22, 72.26 ± 8.77, and 70.46 ± 9.58 mitochondria per µm(3) and 678 ± 147, 245 ± 199, and 346 ± 38 × 10(6) cells, respectively. However, the largest diameters of the large luteal cells were increased in superovulated and control cows versus stimulated ones (32.32 ± 0.06, 31.59 ± 0.81, and 29.44 ± 0.77 µm; P < 0.0001). In contrast, the total number of small luteal cells was increased in superovulated cows (1456 ± 268, 492 ± 181, and 822 ± 461 × 10(6), P < 0.05). In conclusion, there were indications of cellular changes related to increased hormonal production (stimulatory treatment) and increased CL volume (superovulatory treatment).

Global gene expression in the bovine corpus luteum is altered after stimulatory and superovulatory treatments.

Equine chorionic gonadotrophin (eCG) has been widely used in superovulation and artificial insemination programmes and usually promotes an increase in corpus luteum (CL) volume and stimulates progesterone production. Therefore, to identify eCG-regulated genes in the bovine CL, the transcriptome was evaluated by microarray analysis and the expression of selected genes was validated by qPCR and western blot. Eighteen Nelore crossbred cows were divided into control (n=5), stimulated (n=6) and superovulated groups (n=7). Ovulation was synchronised using a progesterone device-based protocol. Stimulated animals received 400 IU of eCG at device removal and superovulated animals received 2000 IU of eCG 4 days prior. Corpora lutea were collected 7 days after gonadotrophin-releasing hormone administration. Overall, 242 transcripts were upregulated and 111 transcripts were downregulated in stimulated cows (P ≤ 0.05) and 111 were upregulated and 113 downregulated in superovulated cows compared to the control animals (1.5-fold, P ≤ 0.05). Among the differentially expressed genes, many were involved in lipid biosynthesis and progesterone production, such as PPARG, STAR, prolactin receptors and follistatin. In conclusion, eCG modulates gene expression differently depending on the treatment, i.e. stimulatory or superovulatory. Our data contribute to the understanding of the pathways involved in increased progesterone levels observed after eCG treatment.

Potencial envolvimento da adiponectina e seus receptores na modulação da esteroidogênese em corpo lúteo de cadelas ao longo do diestro / 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 17β-estradiol and progesterone, as well as the expression of a gene target of the system, the PPAR-γ. 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-γ 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 17β-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-γ 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-γ, participating in the physiological mechanism of development, maintenance, activity and luteal regression in bitches.

Análise comparativa da origem do plexo branquial de catetos (Tayassu tajacu) / Comparative analysis of the origin of the brachial plexus of the collared peccary (Tayassu tajacu)

O cateto (Tayassu tajacu) pertence à familia Tayassui-dae e é caracterizado por "colar" de pêlos brancos ao redor do pescoço que se estende bilateralmente cranialmente aos ombros. Pode ser encontrado do sudoeste dos Estados Unidos da América até a Argentina. Na literatura verificou-se a falta de dados a respeito da anatomia funcional do cateto especialmente trabalhos que envolvem a anatomia do plexo braquial. Visando elucidar o comportamento do plexo braquial do cateto e com a finalidade de contribuir para o desenvolvimento da anatomia comparada, realizou-se esta pesquisa. Utilizou-se 30 animais de idades diferentes (13 fêmeas e 17 machos) provenientes do Centro de Multiplicação de Animais Silvestres, Universidade Federal Rural do Semi-árido, Mossoró, Rio Grande do Norte. Após o abate, realizou-se a dissecação bilateral dos plexos braquiais e registraram-se os resultados através de desenhos esquemáticos e as disposições agrupadas em tabelas para subseqüente análise estatística e obtidas as freqüências percentuais. Observou-se que o plexo braquial de catetos é resultado das comunicações estabelecidas, principalmente, entre os ramos ventrais dos três últimos nervos espinhais cervicais (C6, C7 e C8) e dos dois primeiros nervos espinhais torácicos (T1 e T2), tendo uma contribuição do quarto e quinto nervos cervicais em 16,67 por cento e 50,00 por cento dos casos, respectivamente. Em 40,00 por cento das dissecações a formação do plexo, mais freqüente, foi do tipo C6, C7, C8, T1 e T2. Os principais nervos derivados do plexo braquial dos catetos e suas respectivas origens foram: nervo supraescapular (C6 e C7), nervo subscapular (C5, C6 e C7 ou C6 e C7), nervo axilar (C6 e C7), nervo músculocutâneo (C7 e C8), nervo mediano (C7, C8, T1 e T2), nervo ulnar (C8, T1 e T2), nervo radial (C8, T1 e T2), nervos peitorais craniais (C7) e caudais (C7 e C8), nervo toracodorsal (C6, C7 e C8), nervo torácico longo (C7 e C8) e nervo torácico lateral (C8, T1 e T2).

Collared peccary (Tayassu tajacu) belongs to the Tayassuidae family, characterized by a "collar" of white hairs that cross behind the neck and extend bilaterally in front of the shoulders. It can be found from south-western United States to Argentina. In the literature a shortage of data is verified regarding the functional anatomy of the collared peccaries, especially of studies that involve the anatomy of the brachial plexus. To elucidate the behavior of this plexus of collared peccaries and with the purpose to contribute for the development of compared anatomy, this study was accomplished. Thirty animals of different ages were used (17 males and 13 females) coming from the Wild Animal Multiplication Center of the "Universidade Federal Rural do Semiárido" Mossoró, Rio Grande do Norte, Brazil. After slaughter bilateral dissection of the brachial plexuses took place, and the results were registered in schematic drawings and the dispositions grouped in tables for subsequent statistical analysis based on the percentile frequency. It was found that the Plexus brachialis of collared peccaries is the result of established communications, mainly among the Rami ventrales of the last three cervical nerves and of the first two thoracic nerves, having a contribution of the fourth and fifth cervical nerves in 16.67 percent and 50.00 percent of the cases, respectively. In 40.00 percent of the dissections the most frequent plexus was of the type C6, C7, C8, T1 and T2. The main nerves derived from brachial plexus of the collared peccaries and its respective origins had been: Nervus suprascapularis (C6, C7), Nn. subscapulares (C5, C6 e C7 or C6 e C7), N. axillaris (C6, C7), N. musculocutaneus (C7, C8), N. medianus (C7, C8, T1, T2), N. radialis (C8, T1, T2), N. ulnaris (C8, T1, T2), cranialis (C7), and caudalis (C7, C8) Nn. pectorales , N. thoracodorsalis (C6, C7, C8), N. thoracicus longus (C7, C8), and N. thoracicus lateralis (C8, T1, T2).

Análise comparativa da origem do plexo branquial de catetos (Tayassu tajacu) / Comparative analysis of the origin of the brachial plexus of the collared peccary (Tayassu tajacu)

O cateto (Tayassu tajacu) pertence à familia Tayassui-dae e é caracterizado por "colar" de pêlos brancos ao redor do pescoço que se estende bilateralmente cranialmente aos ombros. Pode ser encontrado do sudoeste dos Estados Unidos da América até a Argentina. Na literatura verificou-se a falta de dados a respeito da anatomia funcional do cateto especialmente trabalhos que envolvem a anatomia do plexo braquial. Visando elucidar o comportamento do plexo braquial do cateto e com a finalidade de contribuir para o desenvolvimento da anatomia comparada, realizou-se esta pesquisa. Utilizou-se 30 animais de idades diferentes (13 fêmeas e 17 machos) provenientes do Centro de Multiplicação de Animais Silvestres, Universidade Federal Rural do Semi-árido, Mossoró, Rio Grande do Norte. Após o abate, realizou-se a dissecação bilateral dos plexos braquiais e registraram-se os resultados através de desenhos esquemáticos e as disposições agrupadas em tabelas para subseqüente análise estatística e obtidas as freqüências percentuais. Observou-se que o plexo braquial de catetos é resultado das comunicações estabelecidas, principalmente, entre os ramos ventrais dos três últimos nervos espinhais cervicais (C6, C7 e C8) e dos dois primeiros nervos espinhais torácicos (T1 e T2), tendo uma contribuição do quarto e quinto nervos cervicais em 16,67 por cento e 50,00 por cento dos casos, respectivamente. Em 40,00 por cento das dissecações a formação do plexo, mais freqüente, foi do tipo C6, C7, C8, T1 e T2. Os principais nervos derivados do plexo braquial dos catetos e suas respectivas origens foram: nervo supraescapular (C6 e C7), nervo subscapular (C5, C6 e C7 ou C6 e C7), nervo axilar (C6 e C7), nervo músculocutâneo (C7 e C8), nervo mediano (C7, C8, T1 e T2), nervo ulnar (C8, T1 e T2), nervo radial (C8, T1 e T2), nervos peitorais craniais (C7) e caudais (C7 e C8), nervo toracodorsal (C6, C7 e C8), nervo torácico longo (C7 e C8) e nervo torácico lateral (C8, T1 e T2).

Collared peccary (Tayassu tajacu) belongs to the Tayassuidae family, characterized by a "collar" of white hairs that cross behind the neck and extend bilaterally in front of the shoulders. It can be found from south-western United States to Argentina. In the literature a shortage of data is verified regarding the functional anatomy of the collared peccaries, especially of studies that involve the anatomy of the brachial plexus. To elucidate the behavior of this plexus of collared peccaries and with the purpose to contribute for the development of compared anatomy, this study was accomplished. Thirty animals of different ages were used (17 males and 13 females) coming from the Wild Animal Multiplication Center of the "Universidade Federal Rural do Semiárido" Mossoró, Rio Grande do Norte, Brazil. After slaughter bilateral dissection of the brachial plexuses took place, and the results were registered in schematic drawings and the dispositions grouped in tables for subsequent statistical analysis based on the percentile frequency. It was found that the Plexus brachialis of collared peccaries is the result of established communications, mainly among the Rami ventrales of the last three cervical nerves and of the first two thoracic nerves, having a contribution of the fourth and fifth cervical nerves in 16.67 percent and 50.00 percent of the cases, respectively. In 40.00 percent of the dissections the most frequent plexus was of the type C6, C7, C8, T1 and T2. The main nerves derived from brachial plexus of the collared peccaries and its respective origins had been: Nervus suprascapularis (C6, C7), Nn. subscapulares (C5, C6 e C7 or C6 e C7), N. axillaris (C6, C7), N. musculocutaneus (C7, C8), N. medianus (C7, C8, T1, T2), N. radialis (C8, T1, T2), N. ulnaris (C8, T1, T2), cranialis (C7), and caudalis (C7, C8) Nn. pectorales , N. thoracodorsalis (C6, C7, C8), N. thoracicus longus (C7, C8), and N. thoracicus lateralis (C8, T1, T2).

Produção de progesterona in vitro pelas células do corpo lúteo bovino ao longo da gestação / In vitro progesterone production from bovine corpus luteum throughout gestation

O presente trabalho foi desenvolvido para testar a hipótese de que células luteínicas bovinas em cultivo, provenientes dos três terços de gestação, comportam-se da mesma maneira que células in vivo em relação à produção de P4. Foram coletadas amostras de corpos lúteos (CL) de 90 (n=3), 150 (n=3) e 210 (n=3) dias de gestação obtidos em abatedouro. Sob condições assépticas, as células foram mecanicamente dispersas e cultivadas em placas de 96 poços. Após 24 horas de cultivo foram feitas a lavagem dos poços e a adição do precursor pregnenolona. Os tratamentos foram realizados em octuplicata para cada tempo de tratamento (24, 48 e 96 horas) com três repetições de cada período gestacional. As amostras de meio de cultura e as células foram coletadas 24, 48 e 96 horas após adição do precursor e acondicionadas em freezer a -20ºC até o processamento. A progesterona foi dosada através de radioimunoensaio e o conteúdo protéico pelo método de Lowry. Os resultados foram analisados estatisticamente e considerados diferentes quando p<0.05. Foi observada maior produção de P4 aos 90 dias de gestação (35,277±0,075), posterior decréscimo aos 150 dias (28,820±0,231) e novo aumento aos 210 dias (32,777±0,099). A produção de P4 em células cultivadas por 24 horas foi maior (p<0,05) em células oriundas do grupo de 90 dias (2,912±0,047) quando comparado a 150 (2,669±0,137) e 210 dias (2,741±0,088). As 48 e 96 horas de cultivo, células luteínicas bovinas de 90 dias produziram mais P4 que células de 210 dias (2,934±0,029 e 2,976±0,121 respectivamente x 2,760±0,059 e 2,695±0,149, respectivamente; p<0,05), que por sua vez produziram mais do que células de 150 dias (2,334±0,084 para 48 horas e 2,205±0,136 para 96 horas). Aos 150 dias de gestação a produção de progesterona apresentou diminuição gradativa ao longo das 96 horas de cultivo. Essas diferenças podem ser explicadas pela expressão gênica diferencial de enzimas ou também de fatores presentes na cascata esteroidogênica...

The aim was to test the hypothesis that cultivated bovine luteal cells from three different thirds of pregnancy behave the same way as in vivo luteal cells relative to P4 production. Corpus luteum samples from days 90 (n=3), 150 (n=3) and 210 (n=3) of pregnancy were obtained at a local slaughterhouse. Under aseptic conditions cells were mechanically dispersed and cultivated in a 96 wells-plate. After 24 hours of culture, cells were washed and the precursor pregnenolone was added. Experiments were conducted eight times for each studied time period (24, 48 and 96 h) and three times for each gestational age. Culture medium and cells were collected after 24, 48 and 96 hours of precursor addition and kept frozen at -20ºC until processing. Progesterone was measured by RIA and protein content by Lowry's method. Results were statistically analyzed and considered different when p <0.05. A higher P4 production was observed on day 90 of gestation (35.277±0.075), then this production was decreased at day 150 (28.820±0.231) and increased again at day 210 (32.777±0.099). After 24 hours of culture, luteal cells P4 production reached maximum values in the group of 90 days (2.912±0.047) when compared to 150 (2.669±0.137) and 210 days (2.741±0.088). At 48 and 96 hours of culture, bovine luteal cells from day 90 of gestation produced more P4 than cells from day 210 (2.934±0.029 and 2.976±0.121 respectively x 2.760±0.059 and 2.695±0.149, respectively; p<0.05), which in turn, produced more P4 than cells from day 150 (2.334±0.084 for 48 h and 2.205±0.136 for 96 h). Luteal cells from day 150 of gestation presented a decreasing P4 production throughout the 96 hours of culture. These differences could be explained by differential gene expression of enzymes and/or factors belonging to the esteroidogenic cascade in accordance to the gestational period. The established luteal cell culture model could be used for further functional studies once P4 secretion pattern...

Immunohistochemical localization of VEGF and its receptors in the corpus luteum of the bitch during diestrus and anestrus.

The corpus luteum (CL) is a temporary endocrine gland, whose life span depends on the interaction of luteotrophic and luteolytic factors. Since development and maintenance of CL is based on angiogenesis, angiogenic growth factors may play a role in CL-function of the bitch, as described for other species. The aim of this study was to detect the presence of the vascular endothelial growth factor (VEGF) system in the bitch CL throughout diestrus and early anestrus. For that purpose, blood samples from 24 bitches were collected and analyzed for progesterone to determine ovulation time and the animals were subjected to ovariosalpingohysterectomy 10, 20, 30, 40, 50, 60 or 70 days after ovulation. The corpora lutea were fixed in formalin and embedded in Paraplast resin. Five micrometers sections were submitted to standard immunohistochemistry protocol using three primary antibodies (SC-315, SC-316 and VG76e) for detection of kinase domain region (KDR), fms-like tyrosine kinase 1 (Flt-1) and VEGF, respectively. The VEGF system expression could be detected in all diestrus stages in endothelial as well as luteal cells (responsible for blood vessel formation and progesterone production, respectively), indicating time dependent changes: immunostaining tended to increase from Day 10 to 50 and to decrease until Day 70 post-ovulation. In the CL of the bitch, structure related cells, like pericytes and stroma cells, expressed it in determined time points of diestrus with little intensity variation. We concluded that VEGF might have a modulatory effect in the CL of the dog acting as paracrine and autocrine factor through its receptors, Flt-1 and KDR.