Selection of dominant follicles in cattle is associated with a deviation in growth rate between the dominant and largest subordinate follicle of a wa

Bovine follicular fluid (FF) constitutes the microenvironment of follicles and includes various biologically active proteins. We performed a study involving 18 healthy non-lactating Holstein cows to determine the protein expression profile of FF at key-stages of follicular development. Follicles were individually aspirated in vivo at pre-deviation (F1∼7.0mm); deviation (F1∼8.5mm); post-deviation (F1∼12.0mm); and pre-ovulatory stages of follicle development, which were confirmed by measurement of follicular estradiol and progesterone concentrations. The FF from nine cows were selected for proteomic analysis. After albumin depletion, triplicates of pooled FF were reduced, alkylated, and digested with trypsin. The resulting peptides were labelled with TMTsixplex and quantified using LC-MS/MS. A total of 143 proteins was identified and assigned to a variety of biological processes, including response to stimulus and metabolic processes. Twenty-two differentially (P < 0.05) expressed proteins were found between stages indicating intrafollicular changes over development, with expected deviation time critical to modulate the protein expression. For instance, high concentrations of follistatin, inhibin, serglycin, spondin-1, fibrinogen, and anti-testosterone antibody were found during early stages of follicular development. In contrast, apolipoprotein H, alpha-2-macroglobulin, plasminogen, antithrombin-III, and immunoglobulins were increased after deviation. Amongst the differentially abundant proteins, 19 were found to be associated with steroidogenesis. Pathway analysis identified proteins that were mainly associated with the acute phase response signaling, coagulation system, complement system, liver/retinoid X receptor activation, and biosynthesis of nitric oxide and reactive oxygen. The differentially expressed proteins provide insights into the size-dependent protein changes in the ovarian follicle microenvironment that could influence follicular function.

In ruminants, uterine pulses of prostaglandin (PG) F_2α characterize luteolysis, while increased PGE₂/PGE₁ distinguish early pregnancy. This study evaluated intrauterine (IU) infusions of PGF_2α and PGE₁ pulses on CL function and gene expression. Cows on day 10 of estrous cycle received 4 IU infusions (every 6h; n = 5/treatment) of saline, PGE₁ (2 mg PGE₁), PGF_2α (0.25 mg PGF_2α), or PGE₁ + PGF_2α. A luteal biopsy was collected at 30 min after third infusion for determination of gene expression by RNA-Seq. As expected, IU pulses of PGF_2α decreased (P < 0.01) P4 luteal volume. However, there were no differences in circulating P4 or luteal volume between Saline, PGE₁, and PGE₁ + PGF_2α indicating inhibition of PGF_2α -induced luteolysis by IU pulses of PGE₁. After third pulse of PGF_2α, luteal expression of 955 genes were altered (FDR < 0.01), representing both typical and novel luteolytic transcriptomic changes. Surprisingly, after third pulse of PGE₁ or PGE₁ + PGF_2α there were no significant changes in luteal gene expression (FDR > 0.10) compared to Saline cows. Increased circulating PGFM (after PGF_2α and PGE₁ + PGF_2α) and PGEM (after PGE₁ and PGE₁ + PGF_2α) demonstrated that PGF_2α and PGE₁ are entering bloodstream after IU infusions. Thus, IU pulses of PGF_2α and PGE₁ allow determination of changes in luteal gene expression that could be relevant to understanding luteolysis and pregnancy. Unexpectedly, by third pulse of PGE₁ there is complete blockade of either PGF_2α transport to the CL or PGF_2α action by PGE₁ resulting in complete inhibition of transcriptomic changes following IU PGF_2α pulses.