O. M. Ohashi

MT3 melatonin binding site, MT1 and MT2 melatonin receptors are present in oocyte, but only MT1 is present in bovine blastocyst produced in vitro

BackgroundMelatonin inclusion into in vitro oocyte maturation (IVM) protocols has been suggested because it possesses a powerful free radical scavenger capability that improves the quality of the oocyte used in in vitro embryo production (IVP). The aim of our study was to investigate the presence of melatonin membrane receptors (MT1and MT2) and MT3, which is the melatonin binding site of NQO2 enzyme, in both oocytes and hatched blastocysts to consider an additional subcellular mechanism responsible for the effects of melatonin on IVP.MethodsThe presence of the high affinity melatonin receptors was investigated through an autoradiographic binding assay, using the non-permeable ligand [125I]-iodomelatonin (17 pM) in embryos. The kind of melatonin site was investigated in oocytes and embryos by immunocytochemistry. In vitro fertilized bovine embryos produced from in vitro maturated oocytes supplemented with melatonin (0.0001 to 1000 nM) were analysed to determine their cleavage and blastocyst formation rates.ResultsThe [125I]-iodomelatonin (17 pM) binding in blastocysts was blocked by pre-incubation with melatonin (30000 nM), showing the presence of the high affinity melatonin receptors. MT1, MT2 and NQO2 immunoreactivity was observed in oocytes. MT1 immunoreactivity was observed in hatched blastocysts, however MT2 and NQO2 were not observed in this embryonic stage. Melatonin (pM) triggered significant difference in both cleavage and blastocysts formation rates.ConclusionsThe high affinity MT1 melatonin receptor must be taking part in IVM events; furthermore it is the first melatonin receptor to appear during bovine embryo development in vitro.

Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Características reprodutivas da paca fêmea (Agouti paca) criada em cativeiro

The objective of this paper was to study the reproductive biology of the Agouti paca raised in captivity. It was observed that the estrous cycle average was 32.5 + 3.7 days, gestation length 148.6 + 4.8 days, parturition interval 224.5 + 52.2 days, and the first post-partum 25.6 + 8.8 days. It was also observed that 55.5% of the females presented two parturitions per year with one young per parturition, of which 44.7% were females and 55.3% were males. The birth weight was 605.9 + 87.8 g for females and 736.7 + 108.4 g for males (P<0.05). Puberty in females occurred between 8 to 12 months; for this, however, more detailed investigations are necessary.

Preservation of goat preantral follicles in saline or coconut water solution

O presente estudo investigou a eficiencia da solucao salina e solucao a base de agua de coco na preservacao de foliculos pre-antrais inclusos em tecido ovariano, em diferentes temperaturas e diferentes tempos de incubacao. No abatedouro, o par ovariano foi dividido em 19 fragmentos; um fragmento ovariano foi imediatamente fixado para histologia classica (controle-tempo zero). Os outros 18 fragmentos ovarianos foram conservados em ambas as solucoes a 4oC, 20oC ou 39oC por 4 h, 12 h ou 24 h. A analise histologica mostrou que a conservacao de fragmentos ovarianos em ambas as solucoes a 4oC por ate 24 h mantem a percentagem de foliculos pre-antrais normais similar aos valores do controle. Ao contrario, a conservacao a 20°C ou 39oC, em ambas as solucoes, reduziu significativamente a percentagem de foliculos pre-antrais normais comparado aos valores do controle, exceto em solucao salina a 20oC por 4 h ou em solucao a base de agua de coco a 20oC por 4 h e 12 h. Em conclusao, esse estudo mostrou que ambas as solucoes podem ser usadas com igual eficiencia para conservar foliculos pre-antrais caprinos a 4°C, independente do tempo de incubacao. No entanto, para conservar foliculos pre-antrais caprinos a altas temperaturas, a solucao a base de agua de coco e recomendada.

Expression of PLIN2 and PLIN3 during oocyte maturation and early embryo development in cattle.

In vitro-produced embryos store high lipid content in cytoplasmic lipid droplets (LD), and reduction or removal of LD has been demonstrated to improve freeze-thaw viability. The Perilipin Adipophilin Tail-interacting Protein of 47 kD (PAT) family of proteins is involved in the formation and regulation of LD in many cell types, but their presence has not been addressed either in cattle oocytes or preimplantation embryos. Therefore, this study aimed to detect the expression of PAT family transcripts (Perilipin-2 [PLIN2] and Perilipin-3 [PLIN3]) in immature and in vitro-matured (IVM) oocytes, and in in vitro-produced embryos at the stages of two to four cells, eight to 16 cells, morulae (MO), and blastocyst (BL). The expression of PLIN3 was downregulated in response to IVM, and PLIN2 was comparatively more expressed than PLIN3 in IVM oocytes (P < 0.001). During the early stages of embryo development, PLIN2 expression reached its peak at the MO stage (P < 0.001) and decreased again at the BL stage. In contrast, PLIN3 was expressed in low levels during the earliest stages of development, slightly upregulated at the MO stage (P < 0.05), and greatly increased its expression at the BL stage (15-fold; P < 0.001). PLIN3 was comparatively more expressed than PLIN2 during embryo culture in most stages analyzed (P < 0.05), except in eight- to 16-cell embryos. These results indicate that PLIN2 might be involved in the maintenance of lipid stocks necessary to support embryo development after fertilization of IVM oocytes. Also, we hypothesize that PLIN3 is the main PAT protein responsible for stabilization of LD formed in consequence of the acute lipid load seen during embryo development. We confirmed the presence of both PLIN2 and PLIN3 proteins in BL at Day 7 using immunocytochemistry: these PAT proteins colocalized with LD stained with BODIPY. PLIN3 seemed to be more ubiquitously spread out in the cytoplasm than PLIN2, consistent with the pattern seen in adipocytes. These findings suggest that both elderly (bigger) and newly formed (smaller) LD, positive for PLIN2 and PLIN3 respectively, coexist in blastocysts. To our knowledge this is the first report showing that transcripts of the PAT family are present in cattle oocytes and embryos.

Derivation of sperm from xenografted testis cells and tissues of the peccary (Tayassu tajacu).

Because the collared peccary (Tayassu tajacu) has a peculiar Leydig cell cytoarchitecture, this species represents a unique mammalian model for investigating testis function. Taking advantage of the well-established and very useful testis xenograft technique, in the present study, testis tissue and testis cell suspensions from immature collared peccaries (n=4; 3 months old) were xenografted in SCID mice (n=48) and evaluated at 2, 4, 6, and 8 months after grafting. Complete spermatogenesis was observed at 6 and 8 months after testis tissue xenografting. However, probably due to de novo testis morphogenesis and low androgen secretion, functionally evaluated by the seminal vesicle weight, a delay in spermatogenesis progression was observed in the testis cell suspension xenografts, with the production of fertile sperm only at 8 months after grafting. Importantly, demonstrating that the peculiar testicular cytoarchitecture of the collared peccary is intrinsically programmed, the unique Leydig cell arrangement observed in this species was re-established after de novo testis morphogenesis. The sperm collected from the xenografts resulted in diploid embryos that expressed the paternally imprinted gene NNAT after ICSI. The present study is the first to demonstrate complete spermatogenesis with the production of fertile sperm from testis cell suspension xenografts in a wild mammalian species. Therefore, due to its unique testicular cytoarchitecture, xenograft techniques, particularly testis cell suspensions, may represent a new and very promising approach to evaluate testis morphogenesis and to investigate spermatogonial stem cell physiology and niche in the collared peccary.

Effect of triiodothyronine on developmental competence of bovine oocytes

Developmental competence of in vitro-matured bovine oocytes is a limiting factor in production of embryos in vitro. Several studies have suggested a potential positive effect of thyroid hormones on cultured oocytes and/or their supporting cells. Therefore, the aim of the present study was to ascertain whether medium supplementation with triiodothyronine (T3) improved subsequent developmental competence of in vitro-matured bovine oocytes. For this purpose, we first documented (using reverse transcription PCR) that whereas bovine cumulus cells expressed both thyroid hormone receptor (TR)-α and TRβ, immature bovine oocytes expressed TRα only. Thereafter, to test the effects of TH on developmental competence, abattoir-derived oocytes were matured in vitro in a medium containing 0, 25, 50, or 100 nM T3 and subjected to in vitro fertilization. Embryo quality was evaluated by assessing cleavage and blastocyst rates, morphological quality, development kinetics, and total cell number on Day 8 of culture. Notably, addition of 50 or 100 nM T3 to the in vitro maturation medium increased (P < 0.05) the rate of hatched blastocysts on the eighth day of culture, as compared with other groups (62.4 ± 11.7, 53.1 ± 16.3, and 32.4 ± 5.3, respectively). Next, the relative expression levels of genes related to embryo quality POU-domain transcription factor (POU5F1) and glucose transporter-1 (GLUT 1) were compared between in vivo- and in vitro-produced blastocysts. On the basis of the previous experiments, IVP embryos originating from oocytes that were matured in vitro in the presence or absence of 50 nM T3 were evaluated. The treatment had no effect (P > 0.05) on gene expression. We concluded that supplementation of bovine oocyte in vitro maturation medium with T3 may have a beneficial effect on the kinetics of embryo development.

Histological study of capuchin monkey (Cebus apella) ovarian follicles

O objetivo do presente trabalho foi obter dados quantitativos e qualitativos da populacao folicular ovariana de femeas de Cebus apella. Foram obtidos 7 ovarios de 4 femeas adultas de C. apella atraves de ovariectomia. Os ovarios foram submetidos a preparacao para histologia otica de rotina. O numero de foliculos pre-antrais e antrais por ovario foi estimado utilizando o Metodo Fracionador. Os foliculos pre-antrais foram classificados em primordial, transicao, primario e secundario. Foram considerados foliculos antrais todos aqueles que apresentavam uma cavidade antral. Todos os foliculos contados foram classificados em normais ou degenerados. Com o intuito de acompanhar o desenvolvimento folicular, os diâmetros medios folicular, oocitario e do nucleo do oocito foram determinados. Todos os resultados foram apresentados em Media ± Erro Padrao. A populacao media de foliculos pre-antrais foi de 56.938 ± 21.888 e 49.133 ± 26.896 para os ovarios direito e esquerdo, respectivamente. A percentagem de foliculos pre-antrais estimados normais foi de 80,00 ± 4,95 %. O diâmetro medio folicular variou de 22,0 ± 0,5 µm a 61,2 ± 4,0 µm. No tocante aos foliculos antrais, a populacao media de foliculos normais e degenerados por ovario foi de 60,0 ± 19,0 e 3 ± 1,8 foliculos, respectivamente. O diâmetro medio folicular foi de 514,4 ± 56,6 µm. Para concluir, as informacoes obtidas neste trabalho poderao servir como parâmetro para posteriores estudos in vivo ou in vitro da foliculogenese de primatas nao-humanos neotropicais da especie C. apella.

Biometria testicular, eletroejaculação e características seminais de caititus, Tayassu tajacu Linnaeus, 1758 (Mammalia, Artiodactyla, Tayassuidae) mantidos em cativeiro na Amazônia Oriental

Estudos relacionados a obtencao e avaliacao de semen de Tayassu tajacu sao escassos, sendo necessarias pesquisas a respeito. Os objetivos do estudo foram avaliar a biometria testicular de caititus adultos cativos, testar a eficiencia da eletroejaculacao para obtencao de semen e avaliar suas caracteristicas seminais ao longo do ano. Procedeu-se a eletroejaculacao em oito animais adultos e as amostras de semen colhidas foram avaliadas quanto as caracteristicas fisicas e morfologicas. Os animais tinham testiculo esquerdo com 3,8 ± 0,4 cm X 2,6 ± 0,3 cm e 2,3 ± 0,2 de consistencia, e testiculo direito com 3,8 ± 0,5 cm X 2,7 ± 0,3 cm e 2,3 ± 0,2 de consistencia. A taxa de sucesso nas colheitas foi de 75,21%. O semen possuiu: volume 0,81 ± 0,86 mL, concentracao 137,44 ± 153 x 106 sptz mL-1, pH 7,92 ± 0,73, motilidade 52,66 ± 28,79%, vigor 2,2 ± 0,8, integridade de membrana plasmatica 55,84 ± 28,55%, defeitos maiores 22,87 ± 12,93%, defeitos menores 9,11 ± 5,88% e defeitos totais 31,52 ± 13,81%. Os animais apresentaram simetria testicular, a eletroejaculacao se mostrou eficiente para a obtencao de ejaculados em caititus e as flutuacoes observadas na producao seminal nao foram suficientes para caracteriza-los como animais de reproducao sazonal.

Effects of follicular phase and oocyte–cumulus complexes quality on the protein profile and in vitro oocyte meiosis competence in Cebus apella

OBJECTIVE To study the protein profile of oocytes and cumulus cells from different sized follicles throughout the follicular phase and to asses the ability of oocytes to progress from the dictyate to metaphase II (MII) stage. DESIGN Animal model study. SETTING Five academic basic research laboratories and the National Primate Centre. ANIMAL(S) Eleven normal, cycling capuchin monkey (Cebus apella) females. INTERVENTION(S) Cumulus-oocyte complexes and denuded oocytes were recovered by antral follicle aspiration. MAIN OUTCOME MEASURE(S) Protein profile analysis of denuded or intact oocytes. RESULT(S) The protein profiles of 25 denuded or intact oocytes recovered on days 5 (six denuded, five intact), 7 (four denuded, four intact), or 9 (one denuded, five intact) of the menstrual cycle were analyzed; in a second experiment, 40 intact oocytes were cultured for 24 (n = 20) or 36 hours (n = 20). The oocytes were denuded, fixed, stained, and microscopically examined to reveal the meiotic stage. The protein profile in each compartment within the cumulus-oocyte complex varied along the follicular development with a predominance of low-molecular-weight proteins in both oocyte and cumulus cells at final stages. No differences were found in the protein profile among oocytes pertaining to different sized follicles that were in the same day of the follicular phase. Oocyte MII competence was achieved only after incubation for 36 hours, and the highest maturation rate occurred in those becoming from dominant follicles. CONCLUSION(S) Our study shows, for the first time in a New World primate species, that the proteins contained in oocytes and cumulus cells reach an identical profile in the late follicular phase. This phenomenon could be related to the oocytes ability to progress to the MII stage.