Criopreservação e autotransplante heterotópico de tecido ovariano de gatas domésticas

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, Departamento de Ciências Fisiológicas, 2013.No Brasil, todas as espécies de felinos selvagens estão sob risco de extinção. Desta forma, técnicas de reprodução assistida aplicáveis a tais espécies são recursos valiosos para a conservação das mesmas. O gato doméstico (Felis catus) tem sido um excelente modelo experimental para estudos que visem aplicação em felinos selvagens. O presente trabalho teve como objetivos testar o efeito de diferentes protocolos de criopreservação, além da eficácia do autotransplante ovariano em manter a viabilidade e retomar o desenvolvimento de folículos pré-antrais inclusos em ovários de gatas domésticas. Um teste de criopreservação foi realizado comparando a eficácia de etilenoglicol (EG), dimetilsulfóxido (DMSO) e a associação de ambos (EG+DMSO) na preservação de folículos a aovarianos após descongelamento (1 Etapa). Na 2 Etapa do experimento, cinco gatas foram submetidas à cirurgia de ovariohisterectomia bilateral. Os animais tiveram seis fragmentos de ovário fresco transplantados para o tecido subcutâneo da região dorsal do pescoço.Foram realizadas cinco biopsias (7, 14, 28, 49 e 63 dias). O tecido ovariano foi fixado em Carnoy, processado para histologia clássica e analisado por microscopia de luz. Durante o período de transplante, foram realizadas análises ultrassonográficas para acompanhar o desenvolvimento do tecido ovariano. Na Etapa 1, a criopreservação com DMSO a 1,5 M mostrou-se mais eficaz. Durante as avaliações ultrassonográficas, foi possível identificar a presença de círculos hipoecóicos com diâmetro aproximado de 1 mm, nos animais 3,4 e 5. Tais estruturas correspondiam a folículos antrais. Nas biopsias , foi possível identificar folículos em todos os estágios do desenvolvimento. As porcentagens médias (±DP) de folículos morfologicamente normais encontrados nos fragmentos referentes às biopsias , realizadas nos dias 7, 14, 28, 49 e 63 pós-transplante, foram, respectivamente, 44,36±30,56, 46,95±13,97, 51,41±14,38, 54,80±36,47 e 37,70±31,11. A maioria dos folículos primordiais foi classificada como normal. Nas biopsias dos dias 7 e 14, houve grande concentração de folículos com o citoplasma do ovócito muito acidófilo e com ausência de núcleo (degeneração tipo 1). A partir do dia 14, muitos apresentaram-se com células da granulosa justapostas, que cobriam o local do ovócito, que estava ausente nestas estruturas (degeneração tipo 2). Em conclusão, é possível manter a morfologia folicular após criopreservação de tecido ovariano felino com DMSO a 1,5 M. Também é mantida a viabilidade e funcionalidade de tecido ovariano felino fresco após autotransplante. ______________________________________________________________________________ ABSTRACTIn Brazil, all wild felids species are endangered. This way, assisted reproduction techniques are a valuable resource to keep these species against extinction. The domestic cat (Felis catus) has been an excellent experimental model for studying new techniques to apply to wild felids. The aim of this work was to test different protocols of cryopreservation and the capacity of ovarian autotransplantation to keep the viability and resume preantral follicle development in domestic cats. The cryopreservation test was performed to compare ethylene glycol (EG), dimethyl sulfoxide (DMSO) and the association of both (EG+DMSO) in the preservation of preantral follicles included st nd in ovarian tissue (1 stage). In the 2 stage of the experiment, five cats were submitted to bilateral ovarian hysterectomy. In these animals, six fragments of fresh ovary were immediately transplanted to the subcutaneous tissue of the dorsal neck Five biopsies were performed after 7, 14, 28, 49 and 63 days of transplantation. The ovarian tissue was fixed in Carnoy, processed for classical histology and analysed under light microscopy. During the transplantation period, the ovarian tissue development and localization was monitored by ultrassonographic analysis. In the Stage 1 of the experiment, the cryopreservation with DMSO 1,5 M showed to be more efficient. During the ultrassonographic evaluation it was possible to identify the presence of hypoechoic circles with approximately 2 mm of diameter in the transplanted tissue of animals 3, 4 and 5. These structures corresponded to antral follicles. In the biopsies it was possible to identify follicles in all developmental stages. The mean (±SD) percentages of morphologically normal follicles found in the biopsies at days 7, 14, 28, 49 and 63 post-transplantation were respectively 44.36±30.56, 46.95±13.97, 51.41±14.38, 54.80±36.47 e 37.70±31.11. The majority of the primordial follicles were classified as normal. At days 7 and 14 biopsies there was a great number of follicles with acidophilic cytoplasm and with absent of nucleus (Type 1 degeneration). From day 14 on, a lot of follicles showed juxtaposed granulosa cells covering the place where the oocyte should be (Type 2 degeneration). Both these follicles were classified as degenerated. In conclusion, it is possible to keep the morphology of ovarian follicles after cryopreservation with DMSO 1,5 M. It is possible to keep the viability and function of fresh feline ovarian tissue after autotransplantation

Avaliação biométrica do casco de cavalos de esporte das modalidades hipismo, tambor, laço comprido e pólo

Objetivou-se avaliar, através da biometria, os cascos dos membros anteriores de equinos participantes de provas de Hipismo Clássico, Tambor, Laço Comprido e Pólo. Em cada modalidade foram avaliados 30 indivíduos totalizando 120 animais. As mensurações lineares (cm) incluíram o comprimento dorsal da pinça, comprimentos lateral e medial dos quartos, altura medial e lateral dos quartos, comprimento lateral e medial dos talões, alturas lateral e medial dos talões, comprimento do casco, largura do casco, comprimento da ranilha, largura da ranilha, e as angulares (graus) foram relativas ao ângulo do casco medido pela pinça (podogoniometria), ângulo da quartela, ângulo dos talões, ângulo das paletas. O comprimento da ferradura, circunferência da coroa do casco e peso corporal também foram avaliados. Através da biometria do casco foi possível avaliar os desequilíbrios do membro anterior de animais atletas e os mais comuns foram: quebra do eixo podal, com 96,7% dos animais apresentando essa alteração no membro torácico direito (MTD) e 95,8% no membro torácico esquerdo (MTE); talões contraídos com 95,0% no MTD e 87,6% no MTE. A modalidade com os maiores desequilíbrios foi a de Laço Comprido, seguida pelos animais de Tambor. Foi encontrada uma alta frequência de desequilíbrio médio lateral em todas as modalidades desportivas. Conclui-se que animais utilizados em provas funcionais apresentam uma alta incidência de desequilíbrios podal nos membros torácicos.This study aimed to evaluate, through biometry, the forelimb hoof of horses participating in show jumping, barrel, long rope and polo competitions. Thirty subjects were assessed in relation to each competition (total of 120 animals). The linear measurements (cm) included the dorsal length of the toe; medial and lateral lengths of the quarter; medial and lateral heights of the quarter; lateral and medial lengths of the heel; medial and lateral heights of the heel; hoof length; hoof width; frog length; and frog width. The following angles (°) were measured: toe angle, pastern angle, heel angle and shoulder palette. The length of the horseshoe, coronet circumference and body weight were also assessed. With the use of hoof biometric evaluation was possible to identify the imbalances of forelimb in athletic horses and the most common were broken-backward hoof angle, with 96.7% of the animals showing this in the right forelimb (RFL) and 95.8% in the left forelimb (LFL); and contracted heels, with 95.0% in the RFL and 87.6% in the LFL. The competition type in which greatest numbers of hoof balance abnormalities were shown was the long rope, followed by the barrel. There were high frequencies of medial/lateral imbalance in all the sports. We conclude that animals used in functional tests have a high incidence of hoof balance abnormalities in the forelimbs

Cryopreservation of Human Ovarian Tissue: A Review

BACKGROUND: Cryopreservation of human ovarian tissue has been increasingly applied worldwide to safeguard fertility in cancer patients, notably in young girls and women who cannot delay the onset of their treatment. Moreover, it has been proposed to patients with benign pathologies with a risk of premature ovarian insufficiency. So far, more than 130 live births have been reported after transplantation of cryopreserved ovarian tissue, and almost all patients recovered their ovarian function after tissue reimplantation. SUMMARY: This review aims to summarize the recent results described in the literature regarding human ovarian tissue cryopreservation in terms of methods and main results obtained so far. To cryopreserve human ovarian tissue, most studies describe a slow freezing/rapid thawing protocol, which is usually an adaptation of a protocol developed for sheep ovarian tissue. Since freezing has been shown to have a deleterious effect on ovarian stroma and granulosa cells, various research groups have been vitrifying ovarian tissue. Despite promising results, only 2 babies have been born after transplantation of vitrified/warmed ovarian tissue. Optimization of both cryopreservation strategies as well as thawing/warming protocols is therefore necessary to improve the survival of follicles in cryopreserved ovarian tissue. KEY MESSAGES: Human ovarian tissue cryopreservation has been successfully applied worldwide to preserve fertility in patients with malignant or nonmalignant pathologies that have a detrimental effect on fertility. Human ovarian tissue cryopreservation could also be applied as an alternative to postpone pregnancy or menopause in healthy women. Slow freezing and vitrification procedures have been applied to cryopreserve human ovarian tissue, but both alternatives require optimization

Cryostorage and retransplantation of ovarian tissue as an infertility treatment

While still considered an experimental procedure in most countries, ovarian tissue cryopreservation and transplantation has been increasingly applied worldwide to restore fertility in patients with malignant and non-malignant pathologies with risk of premature ovarian insufficiency. It has yielded more than 130 live births up to now and almost all transplanted patients recovered their ovarian function. This study summarizes ovarian tissue cryopreservation and transplantation indications, procedures, their efficacy and main results and proposes different strategies to improve this strategy. Although the main focus of this study is on ovarian tissue cryopreservation and transplantation as a strategy to restore fertility, we believe that it is also important to discuss other applications for this approach

Immunodetection and quantification of enzymatic markers in theca cells: the early process of ovarian steroidogenesis in human ovary

The association between theca cells (TCs) and granulosa cells is pivotal to steroid biosynthesis in the ovary, but most data on canonical mechanisms of follicle steroidogenesis come from animal models. Indeed, the specificity of human steroidogenesis has not yet been documented in the ovary. Elucidating the cascade of events in human ovarian tissue would advance TC differentiation research, facilitating development of a transplantable engineered ovary

Immunodetection and Quantification of Enzymatic Markers in Theca Cells: The Early Process of Ovarian Steroidogenesis

The association between theca cells (TCs) and granulosa cells is pivotal to steroid biosynthesis in the ovary. During the late secondary follicle stage, TCs form a layer around granulosa cells, after which their steroidogenic function falls under the control of luteinizing hormone (LH) that activates the cAMP signaling pathway via a G protein-coupled receptor. In addition to perilipin-2, a marker for lipid droplets containing esters as substrates for TCs to produce steroidogenic hormones, other essential proteins, like steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5- > 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5- > 4-isomerase type 2, play a role in the cascade after luteinizing hormone-choriogonadotropic hormone receptor (LH/CG-R) occupation by LH. The aim of the present study was to assess expression levels and corresponding amounts of LH/CG-R, perilipin-2 and enzymes involved in the steroidogenic pathway of TCs based on follicle stage. Immunohistochemical analysis of each of these proteins was therefore performed on ovarian samples from nine adult women, most (n = 8) with BRCA1 and/or BRCA2 mutations undergoing prophylactic bilateral oophorectomy. Pictures were taken of the theca layer of secondary, small (3000 μm) antral follicles and corpora lutea at 100X magnification. ImageJ software was used to analyze the surface area and expression intensity of each protein at each stage, known as the staining index. Overall, our data showed that LH/CG-R, perilipin-2 and StAR expression increased in the course of folliculogenesis and luteinization. Similarly, cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5- > 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5- > 4-isomerase type 2 expression were substantially elevated in TCs during folliculogenesis, evidenced by their coordinated action in terms of area covered and expression intensity. This study, conducted for the first time on human ovarian tissue, contributes to localizing and quantifying expression of key steroidogenic proteins at both intracellular and tissue levels. These findings may shed new light on pathological conditions involving the human ovary, such as androgen-secreting tumors of the ovary and other disorders associated with ovarian TCs in patients with polycystic ovary syndrome

Cat ovarian follicle ultrastructure after cryopreservation with ethylene glycol and dimethyl sulfoxide

Ovarian tissue cryopreservation is a promising technique for fertility maintenance. The aim of this study was to compare the morphology of domestic cat ovarian follicles after tissue cryopreservation with ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). Ovaries from healthy adult cats undergoing elective ovariohysterectomy were used. Eight fragments were obtained from each pair of ovaries: two were used as fresh controls; three were submitted to fresh perfusion toxicity test and perfused with M199, 10% fetal calf serum and 0.4% sucrose containing Me2SO 1.5 M, EG 1.5 M or Me2SO 0.75 M + EG 0.75 M; and the remaining three fragments were perfused as described and submitted to slow freezing. After 45 days of cryopreservation, the samples were thawed, fixed and processed for light and transmission electron microscopy (TEM). The percentages of morphologically normal follicles identified by light microscopy were higher in the control group (94.45%) in comparison to the frozen groups (80.56% with EG, 78.7% with Me2SO and 75.87% with EG + Me2SO). The fresh perfused tissue showed no statistical difference compared to control or frozen samples. The TEM analysis showed less damage in the ultrastructure of follicles from the Me2SO group in comparison with the EG and Me2SO + EG groups. According to the morphological analysis, 1.5 M Me2SO is the best cryoprotectant for cryopreservation of domestic cat ovarian tissue regarding the morphology of preantral follicles after thawing. Further studies regarding the viability of these follicles should be performed

Impact of perinatal bisphenol A and 17β estradiol exposure: Comparing hormone receptor response

Hormonal regulation controls mammary gland (MG) development. Therefore some hormone-related factors can disrupt the early phases of MGs development, making the gland more susceptible to long term modifications in its response to circulating hormones. Endocrine disruptors, such as bisphenol A (BPA), are able to cause alterations in hormone receptor expression, leading to changes in the cell proliferation index, which may expose the tissue to neoplastic alterations. Thus, we evaluated the variations in hormone receptor expression in the MG of 6-month old Mongolian gerbils exposed to BPA and 17β estradiol during the perinatal period. Receptors for estrogen alpha (ERα), beta (ERβ), progesterone (PGR), prolactin (PRL-R), and co-localization of connexin 43 (Cx43) and ERα in gerbils were analyzed, and serum concentrations of estradiol and progesterone were assessed. No alterations in body, liver, and ovary-uterus complex weights were observed. However, there was an increase in epithelial ERα expression in the 17β estradiol (E2) group and in PGR in the BPA group. Although immunohistochemistry did not show alterations in ERβ expression, western blotting revealed a decrease in this protein in the BPA group. PRL-R was more present in epithelial cells in the vehicle control (VC), E2, and BPA groups in comparison to the intact control group. Cx43 was more frequent in E2 and BPA groups, suggesting a protective response from the gland against possible malignancy. Serum concentration of estradiol reduced in VC, E2, and BPA groups, confirming that alterations also impacts steroid levels. Consequently, perinatal exposure to BPA and the reference endogenous estrogen, 17β estradiol, are able to increase the tendency of endocrine disruption in MG in a long term manner, since repercussions are observed even 6 months after exposure

Perinatal exposure to bisphenol A impacts in the mammary gland morphology of adult Mongolian gerbils

The endocrine disruptive effects caused by bisphenol A (BPA) are well known. Despite this, to date, evaluation of its long term effects is limited, meaning that there is still much to be unveiled in terms of alterations caused by perinatal exposure to BPA. Our aim was to determine if perinatal exposure to two different doses of BPA causes long term morphological and molecular alteration effects in the mammary gland (MG). We evaluated MG from Mongolian gerbil offspring exposed perinatally (during gestation and lactation) to 50 or 5000 μg/kg/day BPA. At 90 days of age the animals were subjected to a single dose of N-nitroso-N-methylurea in order to mimic a carcinogenic environment. At 6 months of age, animals in estrous were euthanized for morphological evaluation of the MGs. The MG architecture presented considerable changes in terms of detached epithelial cells, inflammation, glandular hyperplasia, and collagen fiber deposition. Furthermore, a higher index of epithelial cell proliferation was detected in comparison to the intact control group. In addition, we verified a higher molecular expression of EZH2 in the vehicle treated group, indicating that corn oil applied alone can alter the expression of this epigenetic biomarker. In conclusion, BPA perinatal exposure promotes significant changes in glandular cytoarchitecture and increases glandular epithelium proliferation rate, leading to the retention of stem-like properties. This event could compromise the fate and differentiation potential of mammary epithelium