Expression of bta-miR-222 and LHCGR in bovine cultured granulosa cells: Impact of follicle deviation and regulation by FSH/insulin in vitro.

Final antral follicle development and future ovulation are mediated by gonadotropin-induced changes with spatio-temporally regulated expression of genes. Here, we aimed to quantify the relative mRNA abundance of bta-miR-222 and its predicted target, LHCGR, in granulosa cells (GCs) from follicles, after follicle deviation, as well as from GCs cultured in vitro with follicle stimulating hormone (FSH) and/or insulin. Thus, to study the impact of follicle deviation, Nelore heifers (n = 10; Bos taurus indicus) were hormonally synchronized and slaughtered 3 days after ovulation. Then, GCs from the dominant follicle (DF) and its respective subordinate follicle (SF) were recovered for RT-qPCR. For in vitro analysis, small follicles (2-5 mm) were dissected from bovine ovaries collected from a local abattoir. The GCs were isolated and cultured in serum-free medium, or treated with insulin (1 ng/mL or 10 ng/mL) alone or in combination with human recombinant FSH (1 ng/mL), for 6 days. Our findings showed that the relative mRNA abundance of LHCGR in GCs was higher in the DF compared to the SF (p = 0.01). Inversely, bta-miR-222 expression was lower in the DF compared to the SF (p = 0.01). Furthermore, GCs cultured with FSH and insulin together resulted in a higher abundance of LHCGR and a lower abundance of bta-miR-222 (p ≤ 0.05) when compared to GCs cultured with insulin alone. In conclusion, we found that the LHCGR upregulation in GCs from the DF is inversely related to bta-miR-222 expression. We also suggest the involvement of FSH in bta-miR-222 suppression in healthy bovine GCs.

Progesterone dose during synchronization treatment alters luteinizing hormone receptor and steroidogenic enzyme mRNA abundances in granulosa cells of Nellore heifers.

The objective was to investigate effects of progesterone (P4) dose on abundance of luteinizing hormone receptor (LHCGR), aromatase (CYP19A1), 3ß-hydroxysteroid dehydrogenase (HSD3B1), and other steroidogenic mRNA transcripts in granulosa cells from dominant follicles. Nellore heifers were assigned to one of six groups: new, first-use controlled internal drug release device (CIDR1) inserted for 5 days (Large-P4-dose-D5; n = 7) or 6 days (Large-P4-dose-D6; n = 8), prostaglandin (PG)F2α administered on D0 and 1 previously-used CIDR (CIDR3) inserted for 5 days (Small- P4-dose-D5; n = 8) or 6 days (Small-P4-dose-D6; n = 8), CIDR1 inserted on D0 and removed plus PGF2α on D5 (Large-P4-dose-proestrus (PE); n = 7), and CIDR3 and PGF2α on D0 and 1, CIDR3 removed plus PGF2α on D5 (Small-P4-dose-PE; n = 7). Duration of P4 treatment (D5 compared to D6) affected abundances of CYP19A1 mRNA transcripts, with there being greater abundances on D6 than D5 (P ≤ 0.05). Heifers treated with the large dose of P4 had a smaller dominant follicle, less serum and intra-follicular estradiol (E2) concentrations (P ≤ 0.05) and lesser LHCGR, CYP19A1, and HSD3B1 transcript abundances (P ≤ 0.05). Heifers treated to induce PE had a larger follicle diameter (P = 0.09), greater intra-follicular E2 concentrations and larger abundances of CYP19A1 mRNA transcript (P ≤ 0.05) than heifers of the D6 group. Overall, treatment with larger doses of P4 resulted in lesser abundances of LHCGR, HSD3B1, and CYP19A1 mRNA transcripts; thus, potentially leading to development of smaller dominant follicles and lesser E2 concentrations.

Molecular and cellular effects of insulin-like growth factor-1 and LongR3-IGF-1 on in vitro maturation of bovine oocytes: comparative study.

Addition effects of insulin-like growth factor-1 (IGF-1) and its synthetic analogue insulin-like growth factor-1 recombinant-3 (LongR3-IGF-1) after in vitro maturation (IVM) of cattle cumulus-oocyte complexes (COCs) were compared and evaluated on meiotic progression, apoptosis and profile genes of oocyte competence (GDF9, BMP15, BAX, BCL2, OOSP1, IGFBP2, IGBFP4 and IGFBP5), and their respective cumulus cells (AREG, EGFR, FSHR, COX2, BAX, BCL2, IGFBP2, IGFBP4 and IGFBP5). The 739 COCs (n = 10 pools) of bovine ovaries were collected, selected and matured with IGF-1 (100 ng/mL), LongR3-IGF-1 (100 ng/mL), and in two control groups with 0.1% polyvinyl alcohol (PVA) or 10% fetal bovine serum (FBS), for 22-24 h. The statistical analysis was performed by a linear mixed effects model, ANOVA and Tukey tests. There was no statistical difference between experimental groups taken into account the meiotic progression and apoptosis (P > 0.05). Nevertheless, there were statistical differences (P ≤ 0.05) among FBS, IGF-1 and LongR3-IGF-1 groups for IGFBP4 gene expression, and among PVA, IGF-1 and LongR3-IGF-1 for COX2 gene expression in cumulus cells. Moreover, statistical difference was found for BCL2 gene expression between IGF-1, FBS and PVA groups and for IGFBP4 gene expression between LongR3-IGF-1, PVA and FBS in oocytes. There was no statistical difference between experimental groups for other genes evaluated. These results showed a good performance of IVM of bovine oocytes in the presence of LongR3-IGF-1 and the possibility of replacement of IGF-1 and FBS.

Partial luteolysis during early diestrus in cattle downregulates VEGFA expression and reduces large luteal cell and corpus luteum sizes and plasma progesterone concentration.

Our objectives were to investigate potential changes in the size of steroidogenic large luteal cells (LLC) during partial luteolysis induced by a sub-dose of cloprostenol in early diestrus and to determine transcriptional variations in genes involved in corpus luteum (CL) functions. Cows were subjected to an Ovsynch protocol, with the time of the second GnRH treatment defined as Day 0 (D0). On D6, cows were randomly allocated into three treatments: Control (2 mL saline, im; n = 10), 2XPGF (two doses of 500 µg of cloprostenol, im, 2 h apart; n = 8) or 1/6PGF (single dose of 83.3 µg of cloprostenol, im; n = 10). Before treatments and every 8 h during the 48-h experimental period, blood samples were collected and CL volumes measured. Furthermore, two CL biopsies were obtained at 24 and 40 h post-treatment. The 1/6PGF treatment caused partial luteolysis, characterized by sudden decreases in plasma progesterone (P4) concentrations, luteal volume and LLC size, followed by increases (to pretreatment values) in P4 and luteal volume at 24 and 40 h post-treatment, respectively. However, at the end of the study, P4, luteal volume and LLC size were all significantly smaller than in Control cows. Temporally associated with these phenotypes, there was a lower mRNA abundance of VEGFA at 24 and 40 h, and ABCA1 at 24 h (P < 0.05). In conclusion, a sudden reduction in CL size during partial luteolysis induced by a sub-dose of PGF2α analog on day 6 of the estrous cycle was attributed to a reduction in LLC size, although these changes did not account for the entire phenomenon. In addition to its involvement in reducing CL size, decreased VEGFA mRNA abundance impaired CL development, resulting in a smaller luteal gland and lower plasma P4 concentrations compared to Control cows.

Testicular hyperthermia reduces testosterone concentrations and alters gene expression in testes of Nelore bulls.

Increased testicular temperature reduces sperm motility, morphology and fertility. Our objectives were to characterize effects of testicular hyperthermia (scrotal insulation) on acute testosterone concentrations and gene expression in Bos indicus testes. Nelore bulls (n = 20), ∼27 mo of age, 375 kg, scrotal circumference >31 cm, with ≥30% motile sperm, were allocated into four groups (n = 5/group): non-insulated (Control) and insulation removed after 12, 24, or 48 h. Immediately after insulation, intratesticular temperatures (needle thermocouples) were coolest in Control bulls and warmest in 48-h bulls (mean ± SEM, 35.28 ± 0.31 vs 38.62 ± 0.57 °C, P < 0.05). Bulls were castrated and testes recovered. Testicular testosterone concentrations were higher in Control versus 48-h bulls (3119 ± 973.3 and 295.5 ± 122.8 ng/g of tissue, respectively, P < 0.05). Total RNA was extracted, reverse transcribed and RT-qPCR done. For STAR, mRNA abundance decreased from Control to 48 h (1.14 + 0.32 vs 0.32 + 0.5, P < 0.05). For BCL2, expression decreased from Control to 24 h (1.00 + 0.07 vs 0.70 + 0.12, P < 0.05), but then rebounded. In addition, GPX1 had a 70% increase (P < 0.05) at 48 h, whereas HSP70 had a 34-fold increase (P < 0.05) at 12 h and 2- and 14-fold increases (P < 0.05) at 24 and 48 h, respectively. HSF1, BAX, P53 and CASP 8 remained unchanged. Downregulation of STAR, critical in androgen production, was consistent with reduced testosterone concentrations, whereas increased GPX1 enhanced testicular antioxidative capability. Huge increases in HSP70 conferred protection again apoptosis and cell destruction, whereas reduced BCL2 promoted apoptosis. These findings provided novel insights into acute tissue responses (testosterone and gene activity) to testicular hyperthermia in B. indicus bulls.

Gene Expression of Fresh and Frozen/Thawed Canine Embryos.

BACKGROUND: Canine embryo cryopreservation and subsequent transfer are relevant in the use of reproductive technologies. OBJECTIVE: The purpose of this study is the identification and quantification of the gene expression BAX and Bcl2, AQP3, Na+/K+ ATPase alpha-1 and beta-1 and LIFr in canine embryos obtained in vivo and after freezing. MATERIALS AND METHODS: For the collection of embryos, the bitches were identified at pro-estrous until the detection of 80-90% superficial cells. After that, they were artificially inseminated with fresh semen. The embryos were collected after ovariohysterectomy. RNA was extracted and amplified, and embryos were randomly distributed into fresh (Fr) and frozen/thawed (Ft) groups. RESULTS: Eighteen blastocysts were collected from three bitches. Genes BAX, AQP3 and LIFr did not differ among the studied groups. CONCLUSION: We suggest, through these results, that the genes BAX, Bcl2, AQP3, Na + / K + ATPase alpha-1 and beta-1 and LIFr were expressed in canine blastocysts collected in vivo and after slow freezing cryopreservation.

Expression of fibroblast growth factor 22 (FGF22) and its receptor, FGFR1B, during development and regression of bovine corpus luteum.

The aim of this study was to determine the expression of fibroblast growth factor 22 (FGF22) in the bovine corpus luteum (CL) and to investigate the effects of in vivo total or partial cloprostenol-induced luteolysis on the mRNA abundance of FGF22 and its receptor, FGFR1B. Corpora lutea at different stages of development were then dissected from abattoir ovaries (n = 10/stage); a portion of the tissue samples was fixed in paraformaldehyde and the remaining samples were homogenized and subjected to total RNA extraction. To assess mRNA abundance of target genes during induced luteolysis, nineteen cows were synchronized and then randomly assigned to a Latin square design as follows: Control; 2 administrations of prostaglandin F2α (PGF2α, total luteolysis; 2 × 250 µg of cloprostenol sodium) and 1/6PGF2α (partial luteolysis; 83.33 µg of cloprostenol sodium). FGF22 and FGFR1B expression levels were measured by RT-qPCR, and FGF22 protein expression was detected by immunohistochemistry. In summary, FGF22 mRNA was detected at all stages of CL development, and FGF22 protein was also detected in luteal tissue. FGF22 mRNA expression was lower at stage IV than at stage III (P < 0.05), and the same pattern was observed in luteal immunoreactivity. Furthermore, cloprostenol-induced luteolysis, both total and partial, increased FGFR1B mRNA abundance in luteal tissue (P < 0.05), but did not affect FGF22 mRNA abundance. In conclusion, these data suggest a potential role for the FGF22-FGFR1B system during development and regression of bovine CL.

Effect of superstimulation on the expression of microRNAs and genes involved in steroidogenesis and ovulation in Nelore cows.

To better understand the impact of ovarian superstimulation on bovine follicular microenvironment, Nelore cows (Bos taurus indicus) were subjected to ovarian superstimulation with follicle stimulating hormone (FSH, n = 10; P-36 protocol) or FSH combined with eCG (n = 10; P-36/eCG protocol). Follicular fluid was analyzed for cholesterol concentration. Granulosa cells were analyzed by RT-qPCR to assess the expression of genes involved in steroidogenic and ovulatory and expression of microRNAs involved in final follicular development and luteinizing hormone/choriogonadotropin receptor (LHCGR) expression. Plasma concentration of estradiol was also measured. Follicular fluid from the P-36 group showed higher concentration of cholesterol than that of control (non-superstimulated) cows. Plasma concentration of estradiol was higher in the P-36/eCG group. Abundance of STAR and FSHR mRNAs were lower in granulosa cells from the P-36/eCG group. In contrast, LHCGR mRNA abundance was higher in superstimulated granulosa cells from the P-36 group and showed a pattern opposite to that of miR-222 expression. Ovarian superstimulation did not affect the expression of other markers (mmu-miR-202-5p, has-miR-873, has-miR-144, and their target genes, CREB, TGFBR2, and ATG7) of antral follicle development. However, the mRNA expression of VEGF pathway components was modulated by P-36 treatment. Taken together, these results demonstrate that superstimulatory protocols modify steroidogenic capacity, increase plasma estradiol, and regulate the abundance of VEGF system, LHCGR mRNA and suppress the expression of miR-222 in bovine granulosa cells.

Lipid profiles of follicular fluid from cows submitted to ovarian superstimulation.

Ovarian superstimulation with exogenous gonadotropins has been extensively used to produce in vivo-derived embryos for embryo transfer in cattle. This process modifies the antral follicle microenvironment and affects oocyte and embryo quality as well the differentiation of granulosa cells. Lipids play significant roles in the cell, such as energy storage, cell structure, and fine-tuning of the physical properties and functions of biological membranes. The phospholipid (PL) contents as well as the effects of superstimulatory treatments on the PL profile of follicular fluid from cows, however, remain unknown. Therefore, to gain insight into the effects of superstimulation with follicle-stimulating hormone (FSH; P-36 protocol) or FSH combined with equine chorionic gonadotropin (eCG; P-36/eCG protocol) on the profile and abundance of PL from cows submitted or not submitted to superstimulatory protocols, were treated with these two superstimulatory protocols. As a control, non-superstimulated cows were only submitted to estrous synchronization. The follicular fluid was aspirated, the remaining cells removed and the follicular fluid stored at -80 °C until extraction. The lipid screening was performed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and this technique allowed the identification of sphingomyelins (SM) and phosphatidylcholines (PC) and phosphoethanolamines (PE). The relative abundance of the ions observed in the three experimental groups was analyzed by multivariate and univariate statistical models. The phospholipid SM (16:0) and PC (36:4) and/or PC (34:1) were less (P < 0.05) abundant in the P-36 group compared to the control or P-36/eCG groups. However, the PC (34:2) was more (P < 0.05) abundant in both group of superstimulated cows compared to the control. In summary, ovarian superstimulation seems to modulate the PL content of bovine follicular fluid with a significant increase in PC (34:2), which jointly with others PC and SM, seems to offer a suitable biomarker involved with reproductive processes successful as ovary superstimulation response and embryo development.

Evidence that fibroblast growth factor 10 plays a role in follicle selection in cattle.

There is evidence that regulation of follicle selection in cattle involves locally produced growth factors. In the present study, we investigated the expression of members of the fibroblast growth factor (FGF) 7 family during follicle deviation. The largest and second largest follicles were recovered during the second day of a synchronised follicle wave and the future dominant and future subordinate follicles were identified based on diameter and cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) mRNA levels in granulosa cells. Theca cells of the future dominant follicle contained less mRNA encoding FGF7 and FGF10 compared with those from the future subordinate follicle 2.5 days after ovulation, before a significant difference between the diameters of the future dominant and future subordinate follicles could be observed, but FGF22 mRNA levels did not change. Levels of mRNA encoding FGF receptors FGFR1B and FGFR2B in theca and granulosa cells, respectively, were lower in the future dominant follicle compared with the future subordinate follicle. Addition of FGF10 to granulosa cells in vitro significantly decreased oestradiol secretion, as well as CYP19A1, FSH receptor (FSHR) and insulin-like growth factor 1 receptor (IGF1R) mRNA abundance, whereas FGF22 had no effect. We conclude that FGF10 and FGFR2B expression is increased in the future subordinate follicle before morphological deviation, which may contribute to follicle selection.

Gene expression profile in heat-shocked Holstein and Nelore oocytes and cumulus cells.

The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.

Localization of angiotensin receptor type 2 in fetal bovine ovaries.

In the ovary, angiotensin II (ANGII) acts through the type 2 receptor (AGTR2) to induce ovulation and may play a role in follicle atresia. In this study, we determined the expression of AGTR2 mRNA and protein during follicle formation in the bovine ovary. Female fetuses at different gestational ages (60, 75, 90, 120, 150 and 210 days) were used for immunolocalization of AGTR2. At day 60, AGTR2 was localized to the cytoplasm of oogonia; from days 75 to 150, during follicle formation and development to secondary stage, AGTR2 immunostaining was weak and irregular, but from day 210 staining became evident in granulosa cells of preantral follicles and in both granulosa and theca cells of small antral follicles. These data differ from those in pigs, in which AGTR2 protein is detected in preantral follicles throughout gestation. Abundance of AGTR2 mRNA in whole ovaries did not change with fetal age. In conclusion, AGTR2 protein is expressed in ovigerous cords in fetal bovine ovaries but not in preantal follicles until the formation of antral follicles. These data suggest important species-specific differences in the expression of AGTR2 in fetal ovaries from polyovulatory and monovulatory animals.

Influence of feeding differentiation on the age at onset of puberty in Brazilian Bergamasca dairy ewe lambs / Influência da diferenciação alimentar na idade à puberdade em cordeiras da raça Bergamácia Brasileira

The objective of the study was to evaluate body growth and age at onset of puberty on lambs fed two specific diets for low and high growth rates. A herd of 20 Brazilian Bergamasca lambs was divided in two groups (n= 10) and kept confined throughout the experimental period, two animals of the same treatment/pen. Two phases were established: Phase 1, from 90 days of age until the onset of puberty; and Phase 2, from puberty onset up to 1 year old. For Phase 1, two distinct diets were formulated, being: Treatment A, which was formulated to obtain an average daily gain of approximately 150g; and Treatment B, for an average daily gain of about 250g. In Phase 2, a balanced, equal diet was provided to both groups. Every 14 days, the animals were weighed and given average daily gain, average daily dry matter intake and body condition score. From the 5th month of age on, in each group, a vasectomized male was used to detect estrus, establishing age at puberty onset and estrus interval for each lamb. Blood samples were collected every 28 days to determine plasma growth hormone concentration. Treatment B lambs gained more weight and had higher body condition score (P<0.05) and there was no difference for age at puberty onset and plasma growth hormone levels (P>0.05) between treatments. It was found that both treatments showed satisfactory performances. Thus, treatment A may be indicated as a reasonable feeding system to achieve positive responses on confined ewe lambs during growth phase.

O objetivo do estudo foi avaliar o crescimento corporal e idade de início da puberdade em cordeiras sob duas dietas específicas para taxas de crescimento baixas e altas. Um rebanho de 20 cordeiras Bergamácia Brasileira foi dividido em dois grupos (n= 10) e mantido confinado em todo período experimental, sendo dispostos dois animais do mesmo tratamento/baia. Duas fases foram estabelecidas: Fase 1, a partir de 90 dias de idade até o início da puberdade, e Fase 2, a partir do início da puberdade até 1 ano de idade. Para a fase 1, duas dietas distintas foram formuladas, sendo: Tratamento A, formulada para obter um ganho de peso diário de aproximadamente 150g; e Tratamento B, para um ganho de peso médio diário de cerca de 250g. Na Fase 2, uma dieta equilibrada idêntica foi fornecida para ambos os grupos. A cada 14 dias, os animais foram pesados e calcularam-se o ganho médio diário, o consumo médio diário de matéria seca e escore de condição corporal. A partir do quinto mês de idade, em cada grupo, um macho vasectomizado foi usado para detectar estro, estabelecendo a idade de início da puberdade e estro para cada cordeira. As amostras de sangue foram coletadas a cada 28 dias para determinar a concentração plasmática de hormônio do crescimento. Cordeiras do tratamento B ganharam mais peso e tiveram maior escore de condição corporal (P<0,05), mas não houve diferença de idade para o início da puberdade e para os níveis plasmáticos de hormônio do crescimento (P>0,05). Verificou-se que os tratamentos A e B apresentaram desempenhos satisfatórios. Assim, o tratamento A pode ser indicado como um sistema de alimentação para alcançar respostas positivas em cordeiras submetidas ao confinamento durante a fase de crescimento.

Shell occupation by the hermit crab Dardanus insignis (Decapoda, Diogenidae) from the north Coast of São Paulo state, Brazil.

The pattern of shell occupation by the hermit crab Dardanus insignis (Saussure, 1858) from the subtropical region of southeastern coast of Brazil was investigated in the present study. The percentage of shell types that were occupied and the morphometric relationships between hermit crabs and occupied shells were analyzed from monthly collections conducted during two years (from January 1998 to December 1999). Individuals were categorized according to sex and gonadal maturation, weighed and measured with respect to their cephalothoracic shield length (CSL) and wet weight (CWW). Shells were measured regarding their aperture width (SAW), dry weight (SDW) and internal volume (SIV). A total of 1086 hermit crabs was collected, occupying shells of 11 gastropod species. Olivancillaria urceus (Roding, 1798) was most commonly used by the hermit crab D. insignis, followed by Buccinanops cochlidium (Dillwyn, 1817), and Stramonita haemastoma (Linnaeus, 1767). The highest determination coefficients (r2 > 0.50, p < 0.01) were recorded particularly in the morphometric relationships between CSL vs. CWW and SAW vs. SIV, which are important indication that in this D. insignis population the great majority the animals occupied adequate shells during the two years analysed. The high number of used shell species and relative plasticity in pattern of shell utilization by smaller individuals of D. insignis indicated that occupation is influenced by the shell availability, while larger individuals demonstrated more specialized occupation in Tonna galea (Linnaeus, 1758) shell.

Shell occupation by the hermit crab Dardanus insignis (Decapoda, Diogenidae) from the north Coast of São Paulo state, Brazil / Ocupação de conchas pelo ermitão Dardanus insignis na costa norte do estado de São Paulo, Brasil

Abstract The pattern of shell occupation by the hermit crab Dardanus insignis (Saussure, 1858) from the subtropical region of southeastern coast of Brazil was investigated in the present study. The percentage of shell types that were occupied and the morphometric relationships between hermit crabs and occupied shells were analyzed from monthly collections conducted during two years (from January 1998 to December 1999). Individuals were categorized according to sex and gonadal maturation, weighed and measured with respect to their cephalothoracic shield length (CSL) and wet weight (CWW). Shells were measured regarding their aperture width (SAW), dry weight (SDW) and internal volume (SIV). A total of 1086 hermit crabs was collected, occupying shells of 11 gastropod species. Olivancillaria urceus (Roding, 1798) was most commonly used by the hermit crab D. insignis, followed by Buccinanops cochlidium (Dillwyn, 1817), and Stramonita haemastoma (Linnaeus, 1767). The highest determination coefficients (r2 > 0.50, p < 0.01) were recorded particularly in the morphometric relationships between CSL vs. CWW and SAW vs. SIV, which are important indication that in this D. insignis population the great majority the animals occupied adequate shells during the two years analysed. The high number of used shell species and relative plasticity in pattern of shell utilization by smaller individuals of D. insignis indicated that occupation is influenced by the shell availability, while larger individuals demonstrated more specialized occupation in Tonna galea (Linnaeus, 1758) shell.

Resumo O padrão de ocupação de conchas pelo ermitão D. insignis (Saussure, 1858) na região subtropical da costa sudeste do Brasil, foi investigada no presente estudo. Foram analisadas a percentual de tipos de conchas que foram ocupados e as relações morfométricas entre os ermitões e conchas ocupadas, a partir de coletas mensais realizadas durante dois anos (de janeiro de 1998 a dezembro de 1999). Os indivíduos foram classificados de acordo com o sexo e maturação, pesados ​​e medidos em relação ao comprimento escudo cefalotoracico (CEC) e peso úmido (CPU). As conchas foram medidas em relação à sua largura de abertura (LAC), peso seco (PSC) e volume interno (VIC). Um total de 1.086 ermitões foram coletados, ocupando conchas de 11 espécies de gastrópodos. Olivancillaria urceus (Roding, 1798) foi a mais utilizada pelo ermitão D. insignis, seguido por Buccinanops cochlidium (Dillwyn, 1817), e Stramonita haemastoma (Linnaeus, 1767). Os maiores coeficientes de determinação (r2 > 0,50, p < 0,01) foram registrados principalmente nas relações morfométricas entre CEC e CPU contra LAC e VIC, que é uma importante indicação de que nesta população de D. insignis a grande maioria dos animais ocupavam conchas adequadas durante os dois anos analisados. O elevado número de espécies de conchas utilizadas ​​e a relativa plasticidade no padrão de ocupação de conchas pelos menores indivíduos de D. insignis indicaram que a ocupação é influenciada pela disponibilidade de conchas, enquanto os indivíduos maiores demonstraram uma ocupação mais especializadas na concha de Tonna galea (Linnaeus, 1758).

Shell occupation by the hermit crab Dardanus insignis (Decapoda, Diogenidae) from the north Coast of São Paulo state, Brazil

Abstract The pattern of shell occupation by the hermit crab Dardanus insignis (Saussure, 1858) from the subtropical region of southeastern coast of Brazil was investigated in the present study. The percentage of shell types that were occupied and the morphometric relationships between hermit crabs and occupied shells were analyzed from monthly collections conducted during two years (from January 1998 to December 1999). Individuals were categorized according to sex and gonadal maturation, weighed and measured with respect to their cephalothoracic shield length (CSL) and wet weight (CWW). Shells were measured regarding their aperture width (SAW), dry weight (SDW) and internal volume (SIV). A total of 1086 hermit crabs was collected, occupying shells of 11 gastropod species. Olivancillaria urceus (Roding, 1798) was most commonly used by the hermit crab D. insignis, followed by Buccinanops cochlidium (Dillwyn, 1817), and Stramonita haemastoma (Linnaeus, 1767). The highest determination coefficients (r2 > 0.50, p 0.01) were recorded particularly in the morphometric relationships between CSL vs. CWW and SAW vs. SIV, which are important indication that in this D. insignis population the great majority the animals occupied adequate shells during the two years analysed. The high number of used shell species and relative plasticity in pattern of shell utilization by smaller individuals of D. insignis indicated that occupation is influenced by the shell availability, while larger individuals demonstrated more specialized occupation in Tonna galea (Linnaeus, 1758) shell.

Resumo O padrão de ocupação de conchas pelo ermitão D. insignis (Saussure, 1858) na região subtropical da costa sudeste do Brasil, foi investigada no presente estudo. Foram analisadas a percentual de tipos de conchas que foram ocupados e as relações morfométricas entre os ermitões e conchas ocupadas, a partir de coletas mensais realizadas durante dois anos (de janeiro de 1998 a dezembro de 1999). Os indivíduos foram classificados de acordo com o sexo e maturação, pesados e medidos em relação ao comprimento escudo cefalotoracico (CEC) e peso úmido (CPU). As conchas foram medidas em relação à sua largura de abertura (LAC), peso seco (PSC) e volume interno (VIC). Um total de 1.086 ermitões foram coletados, ocupando conchas de 11 espécies de gastrópodos. Olivancillaria urceus (Roding, 1798) foi a mais utilizada pelo ermitão D. insignis, seguido por Buccinanops cochlidium (Dillwyn, 1817), e Stramonita haemastoma (Linnaeus, 1767). Os maiores coeficientes de determinação (r2 > 0,50, p 0,01) foram registrados principalmente nas relações morfométricas entre CEC e CPU contra LAC e VIC, que é uma importante indicação de que nesta população de D. insignis a grande maioria dos animais ocupavam conchas adequadas durante os dois anos analisados. O elevado número de espécies de conchas utilizadas e a relativa plasticidade no padrão de ocupação de conchas pelos menores indivíduos de D. insignis indicaram que a ocupação é influenciada pela disponibilidade de conchas, enquanto os indivíduos maiores demonstraram uma ocupação mais especializadas na concha de Tonna galea (Linnaeus, 1758).

Expression of mRNA Encoding the LH Receptor (LHR) and LHR Binding Protein in Granulosa Cells from Nelore (Bos indicus) Heifers Around Follicle Deviation.

The time at which follicles acquire LHR in bovine granulosa cells is the subject of some controversy among researchers. The main objective of the present study was to assess the mRNA expression of LHR and LRBP (mRNA protein binding), a post-transcriptional suppressor of LHR mRNA expression, in granulosa cells from the two largest follicles around the expected time of follicle deviation in Nelore heifers. First, the interval between ovulation and follicle deviation in 20 Nelore heifers was determined (2.3 ± 0.2 days after ovulation). Ovulation was hormonally synchronized, and then, heifers were slaughtered on days 2, 2.5 and 3 after ovulation (before, during and after, respectively, the expected time of follicle deviation), and granulosa cells from the two largest follicles were collected. The mRNA abundance of an LHR fragment common to all isoforms (total LHR) and LRBP was assessed by real-time RT-PCR, and LHR alternative transcripts were assessed by semiquantitative RT-PCR followed by electrophoresis. LHR mRNA expression was not detected before the expected time of deviation. Total LHR mRNA abundance was greater in the largest follicle and increased from day 2.5 to 3. In contrast, LRBP mRNA was detected starting on day 2 and was more expressed in the second largest follicle on days 2.5 and 3. The present data suggest that the expression of LHR mRNA in bovine granulosa cells is established after follicle deviation and that the lower abundance of LRBP mRNA after the expected time of deviation may contribute to greater expression of LHR in the bovine dominant follicle.

Population biology of the commercially exploited shrimp Artemesia longinaris (Decapoda: Penaeidae) in an upwelling region in the Western Atlantic: comparisons at different latitudes.

This study characterized the population biology of the dendrobranchiate penaeid shrimp Artemesia longinaris Spence Bate, 1888, focusing on population structure, sexual maturity, reproductive period and recruitment, and comparing reproductive parameters of a different populations along western South Atlantic. Samples were collected monthly from March, 2008 to February, 2010 in Macaé, northern coast of Rio de Janeiro State, Brazil, a region influenced by the Cabo Frio upwelling. There was a significantly higher percentage of females and with larger sizes than males. Both carapace length and sexual maturity in Macaé were similar to the dimensions found in populations in the South of the continent (Argentina). Reproductive females were present in all months, with main peaks during winter and summer. Recruitment was also continuous, with peaks, usually one to two months after the appearance of reproductive females, after the reduction of the bottom temperature values of water. These data suggest that November to January would be the appropriate months for legal off-season, due to the higher intensity of spawning females and juveniles during this period. The results of this study contribute to the understanding of the biology of A. longinaris, and could also be a reference to monitor this important fishery resource and consequent legal off-season. Furthermore, this population located at the northern limit of the species distribution is a source of highly relevant comparison for population studies in other areas.

Effects of FGF10 on bovine oocyte meiosis progression, apoptosis, embryo development and relative abundance of developmentally important genes in vitro.

Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22 h in TCM-199 supplemented with 0, 2.5, 10 or 50 ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5 ng/ml FGF10 increased (p < 0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50 ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50 ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10 ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.

Prostaglandin receptors (EP2 and EP4) and angiotensin receptor (AGTR2) mRNA expression increases in the oviducts of Nelore cows submitted to ovarian superstimulation.

Many peptides are responsible for the coordination of muscle contraction, secretion and ciliary beating of the oviduct epithelium to allow the transport of gametes and embryos, including vascular endothelial growth factors (VEGF), prostaglandins (PGs), endotelin-1 (ET-1) and angiotensin II (Ang II). The effect of reproductive biotechnologies used to improve embryo yield on oviduct gene expression is poorly understood. Thus, the aim of the present study was to evaluate the effect of ovarian superstimulation on the mRNA expression of the genes encoding the major peptides involved in oviduct contraction in bovine. Therefore, Nelore cows were submitted to P-36 (n=5) or P-36/eCG (n=5) ovarian superstimulatory protocols and a control group of cows was not submitted to any superstimulatory protocol (n=5). The relative expression of VEGF (VEGF, Flk1, Flt1), Ang II (AGTR2, ACE1), ET1 (ET1, ECE1) and PG pathway members (PGES, EP2, EP4, COX1, COX2) was analyzed using real time RT-PCR in each of oviduct segment (infundibulum, ampulla and isthmus). All target genes were expressed in the three segments of the bovine oviduct; however, specific genes were regulated by ovarian superstimulation: EP2 and EP4 receptors mRNA was affected by P-36/eCG protocol, in the ampulla and infundibulum, respectively; and AGTR2 mRNA was up-regulated by both the P-36/eCG and P-36 protocols in the isthmus. The upregulation of EP2, EP4 and AGTR2 expression in the superstimulated cows suggests a suitable effect of FSH and eCG on bovine oviduct physiology, coordinating the contraction in Nelore cows.