Endocrine Mechanisms of Puberty in Heifers: Estradiol Negative Feedback Regulation of Luteinizing Hormone Secretion

The hypothesis that luteinizing hormone (LH) secretion in prepubertal females is responsive to estradiol negative feedback and that decreased feedback occurs as puberty approaches was tested in heifers. In the first experiment, seven heifers were maintained prepubertal by dietary energy restriction until 508 days of age (Day 0). All heifers were placed on a high-energy diet on Day 0 at which time they received no additional treatment (CONT), were ovariectomized (OVX) or were ovariectomized and subcutaneously implanted with estradiol-17β (OVX-E2). This feeding regimen was used to synchronize reproductive state in all heifers. A second experiment was performed with 16 prepubertal heifers using the same treatments at 266 days (Day 0) of age (CONT, OVX and OVX-E2) but no dietary intake manipulation. In both experiments, LH secretion increased rapidly following ovariectomy in OVX heifers. In the initial experiment, LH secretion was maintained at a low level in OVX-E2 heifers until a synchronous rapid increase was noted coincidental with puberty in the CONT heifer. In the second experiment, LH secretion increased gradually in OVX-E2 heifers and attained castrate levels coincidental with puberty in CONT heifers. A gradual increase in LH secretion occurred as puberty approached in CONT heifers. These results indicate that: a) LH secretion in prepubertal heifers is responsive to estradiol negative feedback; and b) estradiol negative feedback decreases during the prepubertal period in beef heifers

Promising System for Selecting Healthy In Vitro–Fertilized Embryos in Cattle

Conventionally, in vitro–fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors—(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage—pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis.

BACKGROUND: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed. RESULTS: Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated. CONCLUSION: The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells

Regulation of Endometrial Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) in the Ewe

Granulocyte macrophage-colony stimulating factor (GM-CSF) increases ovine interferon-tau (oIFNτ) secretion by ovine conceptuses, but endometrial production of GM-CSF has not been characterized. Endometrial GM-CSF expression was evaluated in ovariectomized ewes implanted with estradiol-17β (E2) and/or progesterone (P4) for 14 days, in day 14 cyclic and day 14 pregnant ewes. Relative levels of endometrial GM-CSF mRNA were 3-fold higher in E2- and E2/P4-treated ewes than that of control or P4-treated ovariectomized ewes. Levels of endometrial GM-CSF mRNA for cyclic ewes were similar to E2- and E2/P4-treated ewes, but amounts of GM-CSF mRNA in pregnant ewes were 2-fold higher. GM-CSF concentrations in endometrial culture media, determined by GM-CSF bioassay, for cyclic and E2/P4-treated ovariectomized ewes were 3-fold higher than those of control, E2- and P4-treated ovariectomized ewes; however, amounts of GM-CSF in pregnant ewes were 2-fold higher. Immunoreactive GM-CSF, examined by western blot, was detected in the culture medium from E2/P4-treated ovariectomized, cyclic and pregnant ewes. Luminal and glandular epithelia and stromal regions were determined to be sites of GM-CSF expression by immunohistochemistry and in situ hybridization techniques. Data indicate that combined E2 and P4 treatment of ovariectomized ewes is sufficient to restore GM-CSF expression to the level found in cyclic ewes; however, GM-CSF mRNA and protein in pregnant ewes is 2-fold greater than in ovariectomized or cyclic ewes. These data suggest that the conceptus, in addition to steroids, may play a role in the regulation of endometrial production of GM-CSF

Different Levels of Ovine Interferon-t Gene Expressions Are Regulated Through the Short Promoter Region Including Ets-2 Binding Site

Regulation of interferon-t (IFNt) production, a conceptus secretory protein implicated in the process of maternal recognition of pregnancy, has not been fully elucidated. Among more than 10 ovine IFNt (oIFNt) gene sequences characterized, approximately 75% of oIFNt transcripts expressed in utero is derived from oIFNt-o10 gene and amounts of transcripts from other oIFNt genes such as oIFNt-o8 or oIFNt-o2 are minimal. It was hypothesized that the variation in expression levels exhibited by oIFNt-o10 and oIFNt-o8/-o2 genes was due to differences in the proximal promoter regions of these oIFNt genes. To test this hypothesis, transient transfection experiments with human choriocarcinoma JEG3 cells were executed with deleted and/or mutated 50-upstream regions of these oIFNt genes attached to the chloramphenicol acetyltransferase (CAT) reporter gene. Because only the Ets-2 binding site located in the oIFNt-o10 gene appeared to differentiate the expression levels of these constructs, the 6 base pair (bp) Ets-2 sequence from the oIFNt-o10 gene inserted into the oIFNt-o8/-o2 gene-reporter construct was examined. The insertion of this Ets-2 binding site into the oIFNt-o8/o2-reporter construct failed to increase the degree of transactivation. Rather than this 6 bp sequence, a 22 bp sequence of the proximal promoter region, including the Ets-2 binding site, of the oIFNt-o10 gene was required for oIFNt-o8/-o2-reporter transactivation. By electrophoretic mobility shift assay (EMSA), nuclear protein(s) bound to this 22 bp from the oIFNt-o10 and oIFNt-o8/o2 genes differed. These results suggest that the short promoter region including the Ets-2 binding site, not the Ets-2 binding region itself, may determine different levels of oIFNt gene expressions seen in utero

Effect of Suckling and Ovariectomy on the Control of Luteinizing Hormone Secretion During the Postpartum Period in Beef Cows

Twenty-two mature pluriparous beef cows were randomly assigned to one of six treatments in a 2 x 3 factorial experiment in order to study the role of suckling and ovarian factors on control of the tonic and episodic release of luteinizing hormone (LH). Twelve cows remained intact (INT) and 10 were ovariectomized (OVX) within 4 days following the day of parturition (Day 0). The suckling intensities were nonsuckled (0), suckled once daily for 30 mm (1) and suckled ad libitum by two calves (2). Blood samples were collected at 1 5-mm intervals for 6 h weekly, from Days 6 to 76 postpartum. The postpartum intervals to initiation of ovarian luteal function were 31 ± 3, 41 ± 4 and 67 ± 1 days (X ± SEM) for INT cows with 0, 1 and 2 suckling intensities, respectively. Mean LH concentrations and frequency of LH pulses increased as time of ovulation approached in INT cows. In OVX animals, both mean LH concentrations and frequency of LH pulses increased as time postovariectomy progressed. No differences were detected in mean LH concentrations or frequency of LH pulses between the two suckled OVX groups. Mean LH in the OVX-O cows was greater on Days 13, 20 and 27 postpartum when compared to the respective days in suckled OVX cows. Frequency of LH pulses tended to be lower (

Endocrine Mechanisms of Puberty in Heifers: Estradiol Negative Feedback Regulation of Luteinizing Hormone Secretion

The hypothesis that luteinizing hormone (LH) secretion in prepubertal females is responsive to estradiol negative feedback and that decreased feedback occurs as puberty approaches was tested in heifers. In the first experiment, seven heifers were maintained prepubertal by dietary energy restriction until 508 days of age (Day 0). All heifers were placed on a high-energy diet on Day 0 at which time they received no additional treatment (CONT), were ovariectomized (OVX) or were ovariectomized and subcutaneously implanted with estradiol-17β (OVX-E2). This feeding regimen was used to synchronize reproductive state in all heifers. A second experiment was performed with 16 prepubertal heifers using the same treatments at 266 days (Day 0) of age (CONT, OVX and OVX-E2) but no dietary intake manipulation. In both experiments, LH secretion increased rapidly following ovariectomy in OVX heifers. In the initial experiment, LH secretion was maintained at a low level in OVX-E2 heifers until a synchronous rapid increase was noted coincidental with puberty in the CONT heifer. In the second experiment, LH secretion increased gradually in OVX-E2 heifers and attained castrate levels coincidental with puberty in CONT heifers. A gradual increase in LH secretion occurred as puberty approached in CONT heifers. These results indicate that: a) LH secretion in prepubertal heifers is responsive to estradiol negative feedback; and b) estradiol negative feedback decreases during the prepubertal period in beef heifers