Casie S. Bass

Progesterone secretion by ovine granulosa cells: effects of nitric oxide and plane of nutrition

The aim was to evaluate the effects of nutritional plane on in vitro progesterone (P4) secretion by granulosa (G) cells cultured in the presence or absence of effectors of the nitric oxide (NO) system. Ewes were randomly assigned into three nutritional groups: control (C), overfed (O; 2 × C), or underfed (U; 0.6 × C). Follicular development was induced by FSH injections. On day 15 of the estrous cycle, G cells were isolated and cultured with or without DETA-NONOate (NO donor), L-NAME (NO synthase [S] inhibitor), Arg and (or) LH for 8 h. DETA-NONOate decreased basal and LH-stimulated P4 secretion, and L-NAME increased basal P4 secretion in all groups. In U, Arg decreased LH-stimulated P4 secretion. These data demonstrate that (i) plane of nutrition affects basal P4 secretion by G cells, (ii) the NO donor decreases, NOS inhibitor increases but Arg does not affect basal P4 secretion, and (iii) effects of Arg on LH-stimulated P4 secretion are affected by plane of nutrition in FSH-treated sheep. Thus, plane of nutrition affects G cell function, and the NO system is involved in the regulation of basal and LH-stimulated P4 secretion. The mechanism of the NO system effects on secretory activity of G cells remains to be elucidated.

Expression of progesterone receptor protein in the ovine uterus during the estrous cycle: Effects of nutrition, arginine and FSH

To evaluate expression of progesterone receptor (PGR) AB in follicle stimulating hormone (FSH)-treated or non-treated sheep administered with arginine (Arg) or saline (Sal) fed a control (C), excess (O) or restricted (U) diet, uterine tissues were collected at the early, mid and/or late luteal phases. In exp. 1, ewes from each diet were randomly assigned to one of two treatments, Arg or Sal administration three times daily from day 0 of the first estrous cycle until uterine tissue collection. In exp. 2, ewes were injected twice daily with FSH on days 13-15 of the first estrous cycle. Uterine tissues were immunostained to detect PGR followed by image analysis. PGR were detected in luminal epithelium (LE), endometrial glands (EG), endometrial stroma (ES), myometrium (Myo), and endometrial and myometrial blood vessels. The percentage of PR-positive cells and/or intensity of staining were affected by phase of the estrous cycle, plane of nutrition, and/or FSH but not by Arg. In exp. 1, percentage of PGR-positive cells in LE and EG but not in ES and Myo was greater at the early and mid than late luteal phase, was not affected by plane of nutrition, and was similar in LE and EG. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in LE, EG and Myo, and was the greatest in LE, less in EG, and least in ES and Myo. In exp. 2, percentage of PGR-positive cells in LE, EG, ES and Myo was affected by phase of the estrous cycle, but not by plane of nutrition; was greater at the early than mid luteal phase; and was greatest in LE and EG, less in luminal (superficial) ES and Myo and least in deep ES. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in all compartments but ES, and was the greatest in LE and luminal EG, less in deep EG, and least in ES and Myo. Comparison of data for FSH (superovulated) and Sal-treated (non-superovulated) ewes demonstrated that FSH affected PR expression in all evaluated uterine compartments depending on plane of nutrition and phase of the estrous cycle. Thus, PGR are differentially distributed in uterine compartments, and PGR expression is affected by nutritional plane and FSH, but not Arg depending on phase of the estrous cycle. Such changes in dynamics of PGR expression indicate that diet plays a regulatory role and that FSH-treatment may alter uterine functions.

Angiopoietin expression in ovine corpora lutea during the luteal phase: Effects of nutrition, arginine and follicle stimulating hormone

The aim of this study was to evaluate angiopoietin (ANGPT) 1 and 2, and tyrosine-protein kinase receptor 2 (TIE2) expression in the corpora lutea (CL) of FSH-treated, or non-treated sheep administered arginine (Arg) or vehicle (saline, Sal), and fed a control (C), excess (O) or restricted (U) diet. Ewes from each dietary group were treated with Arg or Sal (experiment 1), and with FSH (experiment 2). Luteal tissues were collected at the early-, mid- and/or late-luteal phases of the estrous cycle. Protein and mRNA expression was determined using immunohistochemistry followed by image analysis, and quantitative RT-PCR, respectively. The results demonstrated that ANGPT1 and TIE2 proteins were localized to luteal capillaries and endothelial cells of larger blood vessels, and ANGPT2 was localized to tunica media of larger blood vessels. TIE2 protein was also present in luteal cells. In experiment 1, ANGPT1 protein expression was greater in O than C during early- and mid-luteal phases, and was greatest during late-luteal phase, less at the mid- and least at the early-luteal phase; 2) TIE2 protein expression was greatest at the mid-, less at the early- and least at the late-luteal phase; 3) ANGPT1 and 2 mRNA expression was greater at the mid- and late- than the early-luteal phase, and TIE2 mRNA expression was greatest at the late-, less at the mid- and least at the early-luteal phase. The ANGPT1/2 ratio was less at the early- than mid- or late-luteal phases. In experiment 2, ANGPT1 protein expression was greater in O during the mid-luteal phase than in other groups, and was greater at the mid- than early-luteal phase. TIE2 protein expression was highest at the mid-, less at the early- and least during the late-luteal phase. ANGPT1 and 2, and TIE2 mRNA expression was higher at the mid- than the early-luteal phase. During mid-luteal phase, ANGPT1 mRNA expression was greater in C than O and U, ANGPT2 was greatest in C, less in O and least in U, and TIE2 mRNA expression was greater in C than O and U. The ANGPT1/2 ratio was higher in U than in any other group. Comparison of FSH vs. Sal treatment effects (experiment 2 vs. experiment 1) demonstrated that FSH affected ANGPT1 and/or -2, and TIE2 protein and mRNA expression depending on luteal phase and/or diet. Thus, expression of ANGPTs and TIE2 in the CL changes during the luteal lifespan, indicating their involvement in luteal vascular formation, stabilization and degradation. Moreover, this study has demonstrated that plane of nutrition and/or FSH treatment affect the ANGPT system, and may alter luteal vascularity and luteal function in sheep.

Lipid droplets in cultured luteal cells in non-pregnant sheep fed different planes of nutrition.

Accumulation of lipid droplets (LD) in luteal cells likely is important for energy storage and steroidogenesis in the highly metabolically active corpus luteum (CL). The objective of this study was to determine the effect of plane of nutrition on progesterone (P4) secretion, and lipid droplet number and size in cultured ovine luteal cells. Ewes were randomly assigned to one of three nutritional groups: control (C; 100% NRC requirements, n=9), overfed (O; 2×C, n=12), or underfed (U; 0.6×C, n=10). Superovulation was induced by follicle stimulating hormone injections. At the early and mid-luteal phases of the estrous cycle, CL were dissected from ovaries, and luteal cells isolated enzymatically. Luteal cells were incubated overnight in medium containing serum in chamber slides. Media were then changed to serum-free and after 24h incubation, media were collected for P4 analysis, and cells were fixed in formalin and stained with BODIPY followed by DAPI staining. Z-stacks of optical sections of large and small luteal cells (LLC and SLC, respectively) were obtained using a laser-scanning microscope. Rendered 3D images of individual LLC and SLC were analyzed for cell volume, and total and individual LD volume, number and percentage of cellular volume occupied by LD by using Imaris software. Concentrations of P4 in serum and media were greater (P<0.05) at the mid than early-luteal phase, and were not affected by nutritional plane. LD total volume and number were greater (P<0.001) in LLC than SLC; however, mean volume of individual LD was greater (P<0.02) in SLC than LLC. In LLC, total LD volume was greater (P<0.02) in O than C and U ewes. In SLC, total LD volume and number was greater (P<0.003) at the mid than early-luteal phase, and percentage of cell volume occupied by LD was greater (P<0.002) in U than C and O ewes. These data demonstrate that both stage of luteal development and nutritional plane affect selected LD measurements and thus may affect luteal functions. Furthermore, these data confirm that LD dynamics differ among parenchymal steroidogenic luteal cell types.