Oscar A. Peralta

Developmental expression of the cellular prion protein (PrPC) in bovine embryos

The mammalian cellular prion protein (PrPC) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrPSc), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about PrPSc conversion and its role in TSEs, the normal function of PrPC has not been elucidated. In adult mammals, PrPC is most abundant in the central nervous tissue, with intermediate levels in the intestine and heart, and lower levels in the pancreas and liver. PrPC is expressed during neurogenesis throughout development, and it has recently been proposed that PrPC participates in neural cell differentiation during embryogenesis. In order to establish the developmental timing and to address the cell‐specific expression of PrPC during mammalian development, we examined PrPC expression in bovine gametes and embryos through gestation Day 39. Our data revealed differential levels of Prnp mRNA at Days 4 and 18 in pre‐attachment embryos. PrPC was detected in the developing central and peripheral nervous systems in Day‐27, 32‐, and ‐39 embryos. PrPC was particularly expressed in differentiated neural cells located in the marginal regions of the central nervous system, but was absent from mitotically active, periventricular areas. Moreover, a PrPC cell‐specific pattern of expression was detected in non‐nervous tissues, including liver and mesonephros, during these stages. The potential participation of PrPC in neural cell differentiation is supported by its specific expression in differentiated states of neurogenesis. Mol. Reprod. Dev. 79:488–498, 2012.

Transvaginal ultrasound-guided cumulus oocyte complexes aspiration and in vitro embryo production in suckled beef and lactating dairy cattle on pasture-based management conditions

This study was conducted to determine the use of repeated transvaginal ultrasound-guided cumulus oocyte complex (COC) aspiration on COC recovery rate, in vitro embryo production (IVP) and subsequent pregnancy rates in Holstein Friesian (HF) and Aberdeen Angus (AA) cows (Experiment 1), and in pregnant and non-pregnant Holstein Friesian cows (Experiment 2). Cycling, non-pregnant HF (n=17) and AA (n=32) cows with 40-70 days postpartum, between 3 and 5 years of age were used in the Experiment 1. All cows were submitted to repeated transvaginal ultrasound-guided COC aspiration twice a week for 5-7 weeks. Cumulus ooctye complexes (COC) were in vitro matured, fertilized and cultured for 8 days. An overall of 100 and 350 embryos from HF and AA cows respectively were cryopreserved using a conventional slow freezing (Experiment 1). A total of 81 and 285 frozen-thawed embryos from HF and AA cows respectively were transferred to recipient cows. Pregnancy diagnosis was performed at 60 and 150 days of gestation using transrectal ultrasonography. In Experiment 2, cycling non-pregnant (n=9) and pregnant (n=8) HF cows were submitted to repeated ultrasound-guided COC aspiration and COC were in vitro matured, fertilized and cultured as in Experiment 1, except that embryos were cryopreserved but not thawed and transferred as described for Experiment 1. The results of this study indicate that COC recovery rate and blastocyts production are affected by the breed of the donor cow. The quality of blastocyts produced from both breed did not differ in terms of pregnancy and calving rates (Experiment 1). The physiologic state of pregnancy did not affect COC recovery rate and blastocysts production per donor/session (Experiment 2). The use of ultrasound-guided COC aspiration and IVP could be a powerful technique to improve the genetic of beef and dairy cattle managed under pasture-based conditions management in the southern Chile.

Endometrial expression of selected transcripts in postpartum of primiparous Holstein cows with clinical and subclinical endometritis

Postpartum endometritis compromises milk production and fertility in high-producing dairy cows. Infection of the endometrium induces an inflammatory response with secretion of cytokines that lead to polymorphonuclear cells (PMN) influx and bacterial clearance. Considering that only a portion of cows with endometritis is eligible for clinical diagnosis, there is an increasing effort for developing reliable tools and protocols for diagnosis of subclinical endometritis. Recent reports have indicated that primiparous cows are at greater risk of uterine infection and primiparous cows with subclinical endometritis produce less milk compared to healthy cows. In the present study, gene expression profiles were compared for selected cytokine and hormone endometrial transcripts in the postpartum of primiparous Holstein cows with clinical and subclinical endometritis. Cows were classified as healthy (no signs of clinical endometritis), cows with subclinical endometritis (PMN<5% in the cytological sample) and cows with clinical endometritis (PMN>5%). Although, cows with clinical endometritis had greater (P<0.05) relative amounts of mRNA for the IL1A, IL6, IL17A, TNFα, PGES and PGHS2 genes compared to healthy cows; no significant differences were detected between clinical and subclinical endometritis groups. Spearman correlation coefficients were positive between relative amounts of gene expression as indicated by amount of these transcripts and PMN percentages and ranged from 0.74 to 0.93 (P<0.05). Relative amounts of cytokine mRNA suggest similar inflammatory response in the endometrium of cows with subclinical and clinical endometritis. Moreover, differential relative amounts of hormone transcripts suggest dysregulation of the luteolytic mechanism and PG synthases but not ERα in cows with endometritis.

Differential expression of GDF-9 and BMP- 15 during follicular development in canine ovaries evaluated by flow cytometry.

Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) play important functions in follicular and oocyte development in many species. This study evaluated the dynamic expression of GDF-9 and BMP-15 in canine follicles cells using flow cytometry analysis. Follicular cells were removed from three sizes of antral follicles (small, medium and large) from ovaries of bitches throughout the estrus cycle. Cells were incubated with anti-human GDF-9 polyclonal and anti-mouse BMP-15 monoclonal antibodies. A size and complexity discriminatory gate was used for the cytometryc analysis in the initial dot plot and, additionally, a CD45 marker for leukocyte and propidium iodide (PI) were used for erythrocyte and debris discrimination. The evidence corroborated the presence of both proteins in canine follicle cells, but these proteins were not expressed equally during follicular development. The results analyzed by ANOVA showed that GDF-9 expression decreased (P<0.05) during follicular growth in anestrus and proestrous/estrous, but increased in diestrus (P<0.05). The expression levels of BMP-15 rose (P<0.05) from small to medium sizes in anestrous without changing at diestrus. Small antral follicles expressed the highest values of GDF-9 at anestrus while only BMP-15 showed higher value in small antral follicles at proestrous-estrus compared to diestrus and anestrus. Both proteins decreased in proestrous/estrous (P<0.05) with increasing follicle size, registering the lowest levels in large follicles. The flow cytometric assay was able to assess GDF-9 and BMP-15 expression in canine follicular cells, showing that these proteins were differentially expressed during follicular development, possibly related to the special features of canine reproduction.

Time exposure period of bovine oocytes to sperm in relation to embryo development rate and quality.

The objective of the study was to determine the effect of different bovine gamete coincubation times on fertilization and embryo development performance. In vitro matured COCs were co-incubated with sperm at a concentration of 1.5 × 106 spermatozoa/ml in TALP medium for 3 hours (T 3, n = 362), 6 hours (T 6, n = 358), or 18 hours (T 18, n = 350). At the end of the coincubation period COCs from times 3 and 6 groups were post-incubated in a new well of fertilization medium without sperm for additional 15 and 12 h, respectively. Cumulus Oocyte Complexes from the T 18 were co-incubated with the sperm suspension for 18 hours. Presumptive zygotes were cultured for 9 days and embryo development was evaluated on days 2, 8, and 9. Thirty blastocysts from each group were stained and total number of nuclei was recorded. The mean (± SEM) percentages of zygotes to develop into ≥2 cell stage were 71.9 ± 5.0; 72.5 ± 5.3 and 81.2 ± 6.1 % for T 3, 6, and 18, respectively, on day 2 and they did not differ (P = .3) among groups. The mean percentage of blastocysts developed on day 8 (25.6 ± 2.8; 24.2 ± 3.3; 28.4 ± 4.2 % for T 3, 6, and 18, resp.) did not differ (P = .4) among groups. The total number of embryonic nuclei was greater (P < .05) for the blastocysts produced from the shortest co-incubation time (T 3).

Tissue localization of GM-CSF receptor in bovine ovarian follicles and its role on glucose uptake by mural granulosa cells

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a multifunctional cytokine implicated in proliferation, differentiation, and activation of several cell types including those involved in hematopoiesis and reproduction. In the present study, the expression of the α- and β-subunit genes of GM-CSF receptor during follicular development in cattle was assessed. The spatial association of α- and β-subunits of GM-CSF with follicle stimulating hormone receptor (FSHR) and 3β-hydroxysteroid dehydrogenase (3β-HSD), and the temporal associations with gene expression of hexose transporters (GLUTs) in granulosa cells of cattle were also evaluated. The effect of GM-CSF on the functionality of hexose transporters was also determined in an in vitro primary culture of granulosa cells. The spatial association of subunits of the GM-CSF receptor with 3β-HSD and FSHR suggests a potential steroidogenic regulation of GM-CSF in granulosa cells. Immunodetection of GLUTs and uptake kinetic assays confirmed expression and functionality of these genes for hexose transporters in granulosa cells of cattle. Treatment of granulosa cells with GM-CSF, FSH or insulin- like growth factor-I (IGF-I) alone increased 2-deoxyglucose (DOG) or 3-0-methylglucose (OMG) uptake; however, when cells were treated with various combination of these factors there were no additive effect. Unexpectedly, the combination of GM-CSF and FSH decreased DOG uptake compared to FSH treatment alone. Thus, the expression pattern of GM-CSF receptor subunit genes during follicle development in cattle and promotion of DOG and OMG uptake in granulosa cells indicate a role for GM-CSF, FSH and/or IGF-I alone in regulating granulosa cell metabolic activity, specifically by promoting glucose uptake.

262 TISSUE-SPECIFIC ANALYSIS OF PRION EXPRESSION IN EARLY BOVINE FETUSES

The prion protein (PrP) is best known for its mis-folded, pathogenic isoform, which is widely regarded as the infectious agent in transmissable spongiform encephalopathies. However, the role of normal, cellular PrP, a host-encoded 29-kD glycoprotein tethered to the cell membrane by a phosphatidyl-inositol glycane (GPI) anchor, is poorly understood. PrP binds copper with high affinity, has antioxidant activity and may play a role in cell adhesion and/or signaling. PrP is expressed in mouse embryos on 6.5 days post-coitum in extra-embryonic tissue and at 13.5 days in the central and peripheral nervous system, intestine, and dental lamina. Our previous data revealed PrP gene expression in bovine embryos throughout pre-implantation embryo development. As part of a larger effort to map the ontogeny of cellular PrP expression in cattle, we sought here to analyze in early bovine fetuses (1) total PrP gene expression by real-time quantitative PCR (QPCR), and (2) tissue-specific PrP expression by immunohistochemistry. Fetuses were obtained from donor cattle bred by artificial insemination (AI; Day 0) and subjected to mid-ventral laparotomy on Days 32 (n = 2) and 39 (n = 2). Immediately upon recovery, one fetus from each stage was placed in RNAlater for RNA isolation and the other fixed in 10% formalin for immunohistochemistry. RNA was isolated using an RNeasy® mini kit (Qiagen, Valencia, CA, USA). cDNA was generated by reverse transcription with random hexamer priming and used the ΔΔcT method for estimation of PrP expression by QPCR. Tissue-specific expression was determined by immunohistochemistry. Formalin-fixed fetuses were embedded in paraffin, sagittally sectioned, dehydrated, and subjected to an unmasking protocol that employed Vectorlab unmasking solution and autoclaving. Tissues were then probed with a primary anti-PrP monoclonal antibody (SAF 32; Cayman Chemical Company, Ann Arbor, MI, USA). Bound primary antibody was detected with a biotinylated horse anti-mouse secondary antibody complexed to horseradish peroxidase using the ABC kit (Vector Laboratories, Burlingame, CA, USA). Probed sections were then counterstained with hematoxolin and eosin. Neighboring sections, processed identically but to which no primary antibody was added, served as controls. PrP gene expression was detected by QPCR at both stages examined and tended to be higher in Day 39 compared to Day 32 fetuses. PrP immunoreactivity was found throughout the central and peripheral nervous systems, ganglia, nerve trunks, and neural cell populations of sensory organs in both Day 32 and Day 39 fetuses. PrP immunolabeling was also observed in the mesonephric kidney, liver, and heart in the Day 39 fetus. At both stages, immunoreactivity was most intense in the nervous system. Thus, PrP is expressed in a tissue-specific pattern in early bovine fetuses. Tissue distribution of fetal PrP expression appears similar to that of adult PrP. Moreover, PrP appears to be expressed in a developmentally regulated fashion in some tissues.