S. S. D. Santos

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.

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.

Morphological and Morphometric Aspects of Crassostrea rhizophorae (Guilding, 1828) Oocytes in Three Stages of the Gonadal Cycle

Crassostrea rhizophorae (Guilding, 1828) es una de las especies de ostras tropicales cultivadas en la costa brasilera, que presenta alto valor comercial, no existiendo informacion sobre las caracteristicas gonadales, durante las fases reproductivas de esta especie. El objetivo de este trabajo fue analizar la morfologia de los ovocitos y de los foliculos gonadales de Crassostrea rhizophorae, en tres fases del ciclo gonadal. Analizando los cortes histologicos de las gonadas y considerando los ovocitos que presentaban nucleo y nucleolo evidentes, fueron determinadas tres fases del ciclo gonadal: gametogenesis inicial, crecimiento y maduracion. En la fase de gametogenesis inicial, el diametro de los foliculos y de los ovocitos era de 180,29 (± 41,91) e 18,66 (± 6,85) µm, respectivamente; tambien identificamos gran cantidad de tejido conjuntivo interfolicular y un numero mayor de ovocitos previtelogenicos. En la fase de crecimiento, el diametro de los foliculos y de los ovocitos era de 218,02 (± 43,19) y 25,92 (± 9,94) µm, respectivamente. Este fase fue caracterizada por una pequena cantidad de tejido conjuntivo interfolicular y predominio de ovocitos vitelogenicos. En la fase de maduracion, el diametro de los foliculos y de los ovocitos era de 298,16 (± 99,24) y 35,27 (± 6,2) µm, respectivamente, existiendo gran numero de ovocitos maduros. De esta manera, concluimos que durante la implantacion del cultivo, Crassostrea rhizophorae tolera la influencia de los factores intrinsecos y extrinsecos y no presenta alteraciones significativas en su actividad reproductiva

Characterization of folliculogenesis and the occurrence of apoptosis in the development of the bovine fetal ovary.

The aim of this research was to perform in situ quantification, morphometry evaluation, and apoptosis analysis of ovarian follicular wall cells in mechanically isolated follicles obtained from ovaries of bovine fetuses (Bos taurus indicus) between 3 and 9 months of age. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The number of isolated follicles increased from 3 months onward (102.5 ± 141.1, mean ± SEM), peaked at 6 months (12855.0 ± 9030.1), and then decreased by 7 months (3208.7 ± 3249.5), consistent with atresia occurring at these stages. Follicular density was greatest at 4 months, consistent with a sudden boost in follicular activity independent of a corresponding increase in ovarian size. Antral follicles were first observed at 5 months. As fetal age increased, there was a tendency for the percentage of primordial and primary follicles to decrease, and the percentage of secondary follicles to increase. However, the high variability (P < 0.05) for all follicle populations up to 5 months of age precluded further interpretation of these results. Oocyte diameter increased from the primordial (23.6 ± 4.4 μm) to the secondary follicular stages (38.0 ± 14.9 μm). Apoptosis was observed in ovaries from all fetal ages analyzed. We concluded that preantral follicles could be isolated from bovine fetuses by 3 months of age, with apoptosis affecting ovarian follicular dynamics throughout fetal life.

The stage of ovarian development affects organ expression of vitellogenin as well as the morphometry and ultrastructure of germ cells in the freshwater prawn Macrobrachium amazonicum (Heller, 1862)

The objective was to characterize vitellogenin expression in the ovary and hepatopancreas, and to describe the morphometry and ultrastructure of oocytes, in the freshwater prawn Macrobrachium amazonicum at various stages of ovarian development. Five ovarian stages were defined: (I) immature, (II) maturing, (III) mature, (IV) spawned, and (V) reorganized. Ovaries and hepatopancreas were analyzed by immunohistochemistry for vitellogenin expression. Vitellogenin expression in both ovary and hepatopancreas was predominantly widespread, beginning at Stage I, peaking at Stage III, and decreasing in Stages IV and V. Characterization of the ovary included measurement of the following germ cell types: oogonia (OG), and previtellogenic (PV), early vitellogenesis (EV), advanced vitellogenesis (AV), and mature (M) oocytes. Mean ± SD diameter of OG and EV oocytes in Stages I (14.2 ± 5.5 and 119.8 ± 15.7 μm) and II (17.9 ± 4.8 and 114.3 ± 34.6 μm), respectively, were significantly different from that in Stages IV (16.6 ± 4.7 and 107.0 ± 24.6 μm) and V (14.4 ± 4.1 and 101.0 ± 25.2 μm). Both scanning and transmission electron microscopy enabled identification of EV, AV and M oocytes based on the presence of a nucleus, and the organization and distribution of yolk in the cytoplasm. In summary, vitellogenesis occurred both in the hepatopancreas and ovary, with the ovary expressing vitellogenin starting as early as Stage I. This process promoted accumulation of yolk and growth of oocytes, thus favoring sexual maturation of females. This knowledge may be applied to improve production of farmed M. amazonicum.

Cinética da maturação nuclear in vitro de oócitos bubalinos

Com o objetivo de avaliar a cinetica da maturacao in vitro de oocitos bubalinos, foram cultivados 1.619 oocitos com cumulus oophorus compacto em quatro diferentes tratamentos: T1 - TCM 199 com 10% de Soro Fetal Bovino (SFB) (meio base); T2 - Meio base e celulas da granulosa (CG); T3 - Meio base, CG, 10 UI de gonadotrofina corionica equina (eCG) e 10 UI de gonadotrofina corionica humana (hCG); T4 - Meio base, 10 UI de eCG, 10UI de hCG e 1 µg de 17b-estradiol / ml, em tempos de 14 a 17h, 20 a 22h, 23 a 25h, 26 a 28h, 29h e 32h de cultivo em estufa de CO2 a 5% e temperatura de 38,5oC. No tratamento 1, observou-se que 81,83%, 69,61%, 65,88%, 60,28%, 64,98% e 67,67% dos ovocitos reiniciaram a meiose. No tratamento 2, as taxas de reinicio da meiose foram de 76,06%, 72,06%, 69,78%, 68,12%,74,97% e 89,96%, no tratamento 3 estes percentuais foram de 83,59%, 78,86%, 77,49%, 82,4%, 72,92% e 81,06% e com o tratamento 4 os indices foram de 95,0%, 91,82%, 93, 17%, 91,65%, 92,09% e 74,99% nos tempos de 14 a 17h, 20 a 22h, 23 a 25h, 26 a 28h, 29h e 32h de cultivo, respectivamente, podendo-se concluir que o meio com maior suplementacao proporciona maior taxa de reinicio da meiose em menor tempo, assim como o aumento no tempo de cultivo pode levar a maiores indices de degeneracao.

Addition of L-arginine to the fertilization medium enhances subsequent bovine embryo development rates

Although L-Arginine (ARG) has been reported as a promising bovine sperm capacitation agent, its effects on embryo development are still poorly understood. Herein, we compared the effects of ARG and/or heparin (HEP) addition to the fertilization medium for bovine oocytes on sperm capacitation and embryo development. We chose 10 mM ARG based on blastocyst development rates in a titration experiment. Addition of ARG and/or HEP to the fertilization medium resulted in similar rates of blastocyst development (P > 0.05). However, when ARG, but not HEP, was combined with a nitric oxide (NO) synthase inhibitor (N-Nitro-L-ARG-methyl ester, 10 mM) blastocyst development was decreased (P < 0.05). To assess the effects on capacitation, bovine sperm were incubated for 0, 3, and 6 hours in fertilization medium containing ARG and/or HEP and/or N-Nitro-L-ARG-methyl esterand acrosomal exocytosis rates were evaluated using fluorescein isothiocyanate conjugated Pisum sativum lectin (FITC-PSA) staining and flow cytometry. With HEP, acrosomal exocytosis rates were highest by 3 hours of incubation; however, by 6 hours, rates were similar for HEP and/or ARG (P > 0.05) and higher than those in control media (P < 0.05). Although both ARG and HEP increased sperm NO production (P < 0.05), combination with L-NAME only precluded acrosomal exocytosis when ARG added alone in the medium (P > 0.05). These results suggest that although both ARG and HEP supported sperm capacitation, only the effects of the former were driven via NO production. Moreover, ARG was also as effective as HEP at improving blastocyst development rates. Therefore, ARG may be used as a low-cost alternative sperm capacitation agent for bovine in vitro embryo production.

Increasing of blastocyst rate and gene expression in co-culture of bovine embryos with adult adipose tissue-derived mesenchymal stem cells

PurposeDespite advances in the composition of defined embryo culture media, co-culture with somatic cells is still used for bovine in vitro embryo production (IVEP) in many laboratories worldwide. Granulosa cells are most often used for this purpose, although recent work suggests that co-culture with stem cells of adult or embryonic origin or their derived biomaterials may improve mouse, cattle, and pig embryo development.Materials and methodsIn experiment 1, in vitro produced bovine embryos were co-cultured in the presence of two concentrations of bovine adipose tissue-derived mesenchymal cells (b-ATMSCs; 103 and 104 cells/mL), in b-ATMSC preconditioned medium (SOF-Cond), or SOF alone (control). In experiment 2, co-culture with 104 b-ATMSCs/mL was compared to the traditional granulosa cell co-culture system (Gran).ResultsIn experiment 1, co-culture with 104 b-ATMSCs/mL improved blastocyst rates in comparison to conditioned and control media (p < 0.05). Despite that it did not show difference with 103 b-ATMSCs/mL (p = 0.051), group 104 b-ATMSCs/mL yielded higher results of blastocyst production. In experiment 2, when compared to group Gran, co-culture with 104 b-ATMSCs/mL improved not only blastocyst rates but also quality as assessed by increased total cell numbers and mRNA expression levels for POU5F1 and G6PDH (p < 0.05).ConclusionsCo-culture of bovine embryos with b-ATMSCs was more beneficial than the traditional co-culture system with granulosa cells. We speculate that the microenvironmental modulatory potential of MSCs, by means of soluble substances and exosome secretions, could be responsible for the positive effects observed. Further experiments must be done to evaluate if this beneficial effect in vitro also translates to an increase in offspring following embryo transfer. Moreover, this study provides an interesting platform to study the basic requirements during preimplantation embryo development, which, in turn, may aid the improvement of embryo culture protocols in bovine and other species.