Effect of progesterone on GnRH-mediated LH release, oocyte quality, and fertility in cattle

The objective was to investigate the effects of progesterone (P4) on luteinizing hormone (LH) release, follicle development, and oocyte competence in cattle. We tested the general hypotheses that: 1) The suppressive effect of P4 on gonadotrophin releasing hormone (GnRH)-mediated LH release can be overcome by increasing GnRH dose or pre-treatment with estradiol (E2); and 2) a shorter period of P4 exposure during the growing phase of the ovulatory follicle improves oocyte competence and fertility after fixed-time artificial insemination or superstimulation in cattle. In the first experiment, heifers (n=22) were treated with 100 or 200 µg of GnRH or pretreated with E2 prior to administration of GnRH during high or low circulating P4 concentrations to characterize LH release (Chapter 2). Increasing the dose of GnRH did not alter LH secretion; however, E2 pretreatment overcame the suppressive effect of high P4 on LH secretion. Cattle with lower (n=11) P4 concentrations had higher circulating LH concentrations than those with higher P4 concentrations (n=11), and tended to have higher ovulation rates. Two experiments were conducted to determine the effect of the duration of P4 exposure during the ovulatory wave on fertility followed fixed-time artificial insemination or superstimulation. In the first experiment (Chapter 3), the dominant follicle was allowed to grow for 3 days (n=181) or 6 days (n=184). Six days of growth resulted in a larger dominant follicle, but in both groups, ovulatory follicles had similar capacities to ovulate and establish pregnancy. In the second experiment (Chapter 4), multiple follicles were allowed to grow for 3 or 6 days by 8 or 14 injections of FSH (at 12-hour intervals). There was no difference between groups for ovulation rate or total ova/embryo recovery rate. Although the 3-day group had higher embryo quality at slaughter (4 days after insemination), further development (7, 9, and 10 days after insemination) did not differ among groups. The effect of FSH starvation following 4 days of FSH treatment (Chapter 4) resulted in loss of ovulatory capability. Overall, a shorter duration of P4 exposure during ovulatory follicle growth did not improve fertility after fixed-time AI or oocyte competence after superstimulation

Transcriptomic difference in bovine blastocysts following vitrification and slow freezing at morula stage.

Cryopreservation is known for its marked deleterious effects on embryonic health. Bovine compact morulae were vitrified or slow-frozen, and post-warm morulae were cultured to the expanded blastocyst stage. Blastocysts developed from vitrified and slow-frozen morulae were subjected to microarray analysis and compared with blastocysts developed from unfrozen control morulae for differential gene expression. Morula to blastocyst conversion rate was higher (P < 0.05) in control (72%) and vitrified (77%) than in slow-frozen (34%) morulae. Total 20 genes were upregulated and 44 genes were downregulated in blastocysts developed from vitrified morulae (fold change ≥ ± 2, P < 0.05) in comparison with blastocysts developed from control morulae. In blastocysts developed from slow-frozen morulae, 102 genes were upregulated and 63 genes were downregulated (fold change ≥ ± 1.5, P < 0.05). Blastocysts developed from vitrified morulae exhibited significant changes in gene expression mainly involving embryo implantation (PTGS2, CALB1), lipid peroxidation and reactive oxygen species generation (HSD3B1, AKR1B1, APOA1) and cell differentiation (KRT19, CLDN23). However, blastocysts developed from slow-frozen morulae showed changes in the expression of genes related to cell signaling (SPP1), cell structure and differentiation (DCLK2, JAM2 and VIM), and lipid metabolism (PLA2R1 and SMPD3). In silico comparison between blastocysts developed form vitrified and slow-frozen morulae revealed similar changes in gene expression as between blastocysts developed from vitrified and control morulae. In conclusion, blastocysts developed form vitrified morulae demonstrated better post-warming survival than blastocysts developed from slow-frozen morulae but their gene expression related to lipid metabolism, steroidogenesis, cell differentiation and placentation changed significantly (≥ 2 fold). Slow freezing method killed more morulae than vitrification but those which survived up to blastocyst stage did not express ≥ 2 fold change in their gene expression as compared with blastocysts from control morulae

Blastocyst development rate (%; number of expanded blastocysts/number of morulae used per group) in unfrozen control, vitrified and slow-frozen bovine morulae.

<p>Each bar represents mean±SEM from five replicates. Different letters (a,b) on bars represent difference (<i>P</i> < 0.05) between groups.</p

Canonical pathway analysis of gene expression in blastocysts developed from vitrified and slow-frozen vs. control morulae, and blastocysts developed from vitrified vs. slow-frozen morulae.

<p>Score ratio (open circles) depicts the number of genes affected in the treatment versus the total number of genes involved in the pathway (y-axis on right side of each figure).</p

Quantification (fold-change; mean±SEM) of mRNA profiles of 7 genes in <i>in vitro</i> produced bovine blastocysts after cryopreservation treatment [Vitrification (VIT) vs. control and slow freezing (SF) vs. control] using qRT-PCR and microarray analyses (n = 3 replicates per morula group).

<p>Black bars represent the differential level of expression of transcripts detected in the microarray analysis, while light grey bars represent the differential level of expression of the same transcripts obtained by qRT-PCR analysis. Asterisks (*) represent difference between gene expressions determined by microarray and qRT-PCR (<i>P</i> ≤ 0.01) analyses. NS = nonsignificant.</p

Upregulated and downregulated transcripts in blastocysts developed from vitrified and slow-frozen vs. unfrozen control (reference) morulae and in vitrified vs. slow-frozen (reference) morulae (<i>P</i> < 0.05).

<p>Transcripts with known functions and novel transcripts are listed separately.</p

Functional analysis of differential gene expression in IVP bovine embryos based on–log (<i>P</i>-value) obtained with Ingenuity^® Pathway Analysis software.

<p>Higher log values relate to higher significance of the functions. Top 10 cellular and molecular functions in each comparison are illustrated. Taller bars are more significant than shorter bars and the dotted line represents the cut-off value for <i>P</i> ≤ 0.05, -log-value = 1.3. Abbreviations: CON–control; SF–slow freezing; VIT–vitrification.</p

Primer sequences used for qRT-PCR.

<p>Primer sequences used for qRT-PCR.</p

Top five upregulated and downregulated genes detected by FlexArray^® analysis in blastocysts developed from vitrified vs. unfrozen control (reference) morulae, slow-frozen vs. unfrozen control (reference) morulae, vitrified vs, slow-frozen (reference) morulae.

<p>Top five upregulated and downregulated genes detected by FlexArray^® analysis in blastocysts developed from vitrified vs. unfrozen control (reference) morulae, slow-frozen vs. unfrozen control (reference) morulae, vitrified vs, slow-frozen (reference) morulae.</p

Functional network of differentially expressed genes in bovine blastocysts following vitrification at morula stage.

<p>All genes involved in this network are part of the matrix-remodeling network. Genes are arranged horizontally in four cell compartments (nucleus, cytoplasm, plasma membrane and extracellular space), based on subcellular location of their gene products. The differences in color intensity of molecules show the degree of up- (red) and down- (green) regulation. The relationship lines between molecules and functions are supported by at least one reference derived from the literature, textbooks, and/or canonical pathways stored in Ingenuity^® Knowledge Base.</p