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Corpora lutea in superovulated ewes fed different planes of nutrition
Accepted Manuscript Corpora lutea in superovulated ewes fed different planes of nutrition A. Kraisoon, D.A. Redmer, C.S. Bass, C. Navanukraw, S.T. Dorsam, V. Valkov, A. Reyaz, A.T. Grazul-Bilska PII:
S0739-7240(17)30108-X
DOI:
10.1016/j.domaniend.2017.08.002
Reference:
DAE 6277
To appear in:
Domestic Animal Endocrinology
Received Date: 16 May 2017 Revised Date:
28 July 2017
Accepted Date: 5 August 2017
Please cite this article as: Kraisoon A, Redmer DA, Bass CS, Navanukraw C, Dorsam ST, Valkov V, Reyaz A, Grazul-Bilska AT, Corpora lutea in superovulated ewes fed different planes of nutrition, Domestic Animal Endocrinology (2017), doi: 10.1016/j.domaniend.2017.08.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Corpora lutea in superovulated ewes fed different planes of nutrition*
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A. Kraisoona, D.A. Redmerb, C.S. Bassb, C. Navanukrawac, S.T. Dorsamb, V. Valkovb,
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A. Reyazb, and A.T. Grazul-Bilskab**
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Thailand;
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Department of Animal Sciences, North Dakota State University, Fargo, ND, USA
Agricultural Biotechnology Research Center for Sustainable Economy (ABRCSE), Khon Kaen
University, Khon Kaen, Thailand.
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Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen,
*A preliminary report of these data was presented at the Western Section of the American
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Society of Animal Sciences meetings in June 2017.
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**Corresponding author. Tel.: +1 701 231 7992; fax: +1 701 231 7590.
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E-mail address: [email protected] (A.T. Grazul-Bilska).
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ABSTRACT The corpus luteum (CL) is an ovarian structure which is critical for the maintenance of reproductive cyclicity and pregnancy support. Diet and/or diet components may affect some
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luteal functions. FSH is widely used to induce multiple follicle development and superovulation.
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We hypothesized that FSH would affect luteal function in ewes fed different nutritional planes.
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Therefore, the aim of this study was to determine if FSH-treatment affects 1) ovulation rate; 2)
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CL weight; 3) cell proliferation; 4) vascularity; 5) expression of endothelial nitric oxide (eNOS)
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and soluble guanylyl cyclase (sGC) proteins; and 6) luteal and serum progesterone (P4)
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concentration in control (C), overfed (O) and underfed (U) ewes at the early- and mid-luteal
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phases. In addition, data generated from this study were compared to data obtained from non-
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superovulated sheep and described by Bass et al. [1]. Ewes were categorized by weight, and
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randomly assigned into nutrition groups: C (2.14 Mcal/kg; n=11), O (2xC; n=12), and U (0.6xC;
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n=11). Nutritional treatment was initiated 60 d prior to d 0 of the estrous cycle. Ewes were
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injected with FSH on d 13-15 of the first estrous cycle, and blood samples and ovaries were
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collected at early- and mid-luteal phases of the second estrous cycle. The number of CL/ewe was
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determined, and CL were dissected and weighed. CL were fixed for evaluation of expression of
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Ki67 (a proliferating cell marker), CD31 (an endothelial cell marker), and eNOS and sGC
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proteins using immunohistochemistry and image analysis. From d 0 until tissue collection, C
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maintained, O gained and U lost BW. The CL number was greater (P < 0.03) in C and O than U.
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Weights of CL, cell proliferation, vascularity, and eNOS but not sGC expression were greater (P
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< 0.001), and serum, but not luteal tissue, P4 concentrations tended to be greater (P = 0.09) at the
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early- than mid-luteal phase. Comparisons of CL measurements demonstrated greater (P < 0.01)
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cell proliferation and serum P4 concentration, but less vascularity at the early and mid-luteal
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phases, and less CL weight at the mid-luteal phase in superovulated than non-superovulated
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ewes; however, concentration of P4 in luteal tissues was similar in both groups. Thus, in
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superovulated ewes, luteal cell proliferation and vascularity, expression of eNOS, and serum P4
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concentration depends on the stage of luteal development, but not diet. Comparison to control
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ewes demonstrated several differences and some similarities in luteal functions after FSH-
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induced superovulation.
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Keywords: Corpora lutea; eNOS; vascularity; FSH; superovulation; sheep
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1. Introduction
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The mammalian corpus luteum (CL), the major source of progesterone (P4) in females, is a transient endocrine gland, which grows, differentiates and regresses during each estrous cycle
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and pregnancy, and plays a major role in reproductive processes [2-7]. Growth, differentiation
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and regression of the CL depend on a balance between luteotropic (e.g., luteinizing hormone)
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and luteolytic (e.g., prostaglandin F2α) factors that subsequently regulate P4 secretion [6,7]. In
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addition, luteal functions may be affected by selected environmental factors, such as nutrition
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[1,8-9]. For example, overfeeding resulted in enhanced serum P4 concentration, and inadequate
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diet (e.g., excess or restriction) altered luteal vascularity, cell proliferation, and mRNA
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expression of selected angiogenic factors in non-pregnant sheep [1,8].
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Dynamic changes within the CL during each estrous cycle include cell proliferation, and
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the establishment and growth of the vascular bed during early- and mid-luteal phases of the
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estrous cycle [1]. The CL consists of several cell types including parenchymal steroidogenic
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small and large luteal cells, vascular cells (endothelial cells, pericytes and smooth muscle cells),
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fibroblasts, immune cells and others [6,7]. It has been suggested that these cell types interact to
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maintain normal luteal function [3,7]. The CL is highly vascularized, and vascular cells comprise
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more than 50% of the total cells in the CL [4]. Vascular growth and function is regulated by
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several angiogenic and growth factors including those associated with production of nitric oxide
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(NO) [1,4,5]. Nitric oxide is a free radical gaseous molecule that is produced in endothelial cells
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by endothelial NO synthase (eNOS)-mediated breakdown of L-arginine [11]. In addition, NO
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diffuses rapidly from endothelial cells into the underlying smooth muscle cells or pericytes
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where it activates soluble guanylate cyclase (sGC). Both, eNOS and sGC are expressed in CL of
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non-pregnant sheep [1,12,13]. Furthermore, the NO system is involved in the regulation of luteal
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functions such as steroid production, angiogenesis, vascularity, apoptosis and luteolysis in
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several species [14-24].
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Follicle stimulating hormone has been widely used to stimulate multiple follicle development and/or superovulation in mammalian species [25-30]. Superovulated models are
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predominantly used for embryo production and to study selected reproductive processes
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including luteal functions in several species [24,31-37]. In sheep and cows, superovulation has
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resulted in greater number of CL and serum P4 concentrations compared to non-treated animals
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[25,27,37-40]. Furthermore, morphology of the CL, and response to LH was similar for
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superovulated and non-superovulated ewes [39,41], but in superovulated cows, expression of
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several genes in luteal tissues was altered, and vascular volume density in CL was increased
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[34,35,37]. However, only very few studies (cited above) evaluated luteal functions in
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superovulated animals. Since superovulation protocols are being used in animal production and
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research, and human medicine [25-30,38-41], and the CL is critical for maintaining normal
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reproductive processes and thus, fertility [6,7], study of luteal functions in superovulated models
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are warranted. Therefore, the aim of this study was to characterize the CL from superovulated
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ewes fed control, excess and restricted diets by determining cell proliferation, vascularity
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(marked by CD31 expression), expression of eNOS and sGC proteins, and serum and luteal P4
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concentrations at the early- and mid-luteal stages of the estrous cycle. These stages correspond to
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the rapid growth and differentiating phases of luteal development [2,3]. Furthermore, luteal
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measurements generated from this study were compared to the same measurements generated
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from non-superovulated ewes in our laboratory [1].
2. Materials and Methods
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2.1. Animals and Experimental Design
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All animal procedures performed were approved by the North Dakota State University (NDSU) Institutional Animal Care and Use Committee (#A12013). The study was initiated
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during the normal breeding season in August and finished in December.
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Non-pregnant, non-lactating Rambouillet ewes between 3 to 5 years of age and of similar
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genetic background were individually penned at the Animal Nutrition and Physiology Center on
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the NDSU campus. Ewes were stratified by body weight (BW) and randomly assigned into one
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of three dietary groups: maintenance-control (C; 100% National Research Council [NRC]
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requirements; 2.4 Mcal of metabolizable energy [ME]/kg BW), overfed (O; 200% NRC
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requirements), or underfed (U; 60% NRC requirements), as previously described [1,8]. Diets
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were initiated 60 d prior to the onset of estrus (d 0). Ewes were fed their individual diets twice
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daily at 0800 and 1500h for the duration of the experiment, and ewes were weighed once weekly.
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Individual rations were adjusted weekly to ensure the proper BW (e.g., C, O and U) was
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achieved at d 0, and maintained throughout the estrous cycle and until completion of the
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experiment. Estrus was synchronized by insertion of a controlled internal drug release (CIDR)
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device for 14 d. Approximately 36 h after removal of the CIDR, ewes were in estrus, which was
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treated as d 0 of the estrous cycle [1,42,43]. Ewes (n = 34) were injected twice daily (morning and evening) with follicle stimulating
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hormone (FSH-P; Sioux Biochemical, Sioux Center, IA, USA) on d 13 to 15 of the first estrous
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cycle (5 mg/injection, 4 mg/injection, or 3 mg/injection, respectively) [43]. This protocol of
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FSH-treatment, reported extensively by our group, has caused multiple ovulations resulting in
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16±0.5 CL per sheep in years 1989-2005 [25]. Initial BW and BCS were similar for all groups
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(55.9 ± 1.5 kg and 2.9 ± 0.1, respectively). Throughout the study, C (n = 11) maintained BW, O
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(n = 11) ewes gained (P < 0.001) 13.1 ± 1 kg and BCS increased (P < 0.01) by 1.0 ± 0.1, and U
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(n = 12) ewes lost (P < 0.001) 7.7 ± 1 kg and BCS decreased (P < 0.01) by 0.7 ± 0.1, similar to
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our previous studies [1,43,44].
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2.2. Tissue and Blood Collection, Immunohistochemistry and Image Analysis Ovaries and blood samples were collected at early- (d 5) and mid- (d 10) luteal phases of the second estrous cycle [43]. Blood samples were centrifuged (20 min at 1,500 g), and serum
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stored at -20˚C until P4 analysis. Number of CL was recorded, and CLs were dissected from
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ovaries and weighed. Randomly selected CL from each ewe was divided into three portions: the
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first portion was fixed in 10% neutral buffered formalin (NBF) for immunohistochemistry, the
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second portion was fixed in Carnoy’s solution for immunohistochemistry, and the third portion
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(approximately 20-50 mg) was immediately frozen on dry ice and stored at -80 ˚C until
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homogenization in PBS (100 mg/1 ml) for evaluation of P4 concentrations.
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Immunohistochemistry was performed as described in detail by Bass et al. [1]. Tissue sections underwent antigen recovery (2100 Retriever, Prestige Medical, Lancashire, England) for
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20 minutes in a 10 mM sodium citrate buffer with 0.05% Tween (pH 6), were washed and
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blocked with 10% normal goat serum for 20 minutes at room temperature, incubated with a
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specific primary antibody, and then with a secondary antibody. Table 1 presents fixatives used,
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tissue thickness, source and dilution of primary and secondary antibodies for all antigens
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immunodetected. Tissue sections stained for CD31, Ki67, eNOS and sGC were cover slipped
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using Prolong Gold with DAPI mounting media (Life Technologies, Eugene, OR, USA).
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Photomicrographs were taken with a Zeiss Imager M2 epifluorescence microscope
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equipped with an AxioCam HRm camera (Zeiss Inc., Thornwood, NY, USA). Images used for
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image analysis were taken from areas where distribution of steroidogenic and accessory cells
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was uniform; areas with connective tissue were avoided. The percentage of the area that
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exhibited positive staining for CD31, eNOS or sGC was evaluated quantitatively with an image
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analysis software package (Image Pro-Plus, Media Cybernetics, Silver Spring, MD) as described
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previously [1]. Labeling index (LI) was calculated as the percentage (%) of proliferating Ki67-
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positive cells out of the total number of cells (DAPI nuclear staining) within the tissue area. For
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each CL, four randomly chosen fields were evaluated in each tissue section. Background
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fluorescence was minimal and was adjusted to the same level for each section by the image
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analysis system. Data are expressed as the percentage of positive staining out of total tissue area
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within each field.
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2.3. Progesterone Analysis
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Progesterone concentration in serum and luteal tissues was determined using a solid phase chemiluminescence, competitive binding immunoassay (Immulite 1000, Siemens, PA,
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USA), as previously described [1,8]. Each sample was run in duplicate. Intra-assay CVs were
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6.1% for serum and 5.5% for luteal P4.
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2.4. Statistical analysis
Data were analyzed statistically using the GLM procedure of SAS 9.2 (Cary, NC, USA).
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The model included plane of nutrition, phase of the estrous cycle, and their interactions. For
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comparison between non-superovulated vs. superovulated ewes, the model included FSH- vs.
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saline-treatment, and interactions. Data presented as percentage were analyzed using Chi square.
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When the F-test was significant (P ≤ 0.05), the differences between specific means were
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separated by using the least significant difference. Data are expressed as mean ± SEM.
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3. Results
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The weights of CL and serum P4 concentrations were greater (P < 0.02-0.09) at midthan early-luteal phase, and were not affected by nutritional plane; therefore, combined data for
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the three nutritional groups are presented in Table 2. The concentrations of P4 in luteal tissues
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were similar (P > 0.1) at early- and mid -luteal phases of the estrous cycle (Table 2). The number
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of CL, and thus ovulation rate, was greater (P < 0.03) in O and C when compared to U (Fig. 1).
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The number of CL ranged from 4-29, 4-27 and 4-22 in C, O and U, respectively. The proportion
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of ewes that did not respond to FSH treatment (the number of CL ≤ 3) was 19%, and it was not
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affected by the plane of nutrition (21, 7 and 27% in C, O and U, respectively).
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Ki67, CD31, eNOS and sGC proteins were immunodetected in CL from early- and midluteal phases of the estrous cycle (Fig. 2 and 3). Ki67 (Fig. 2A and B) was localized to cell
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nuclei, but CD31 (Fig. 2C and D), eNOS (Fig. 2E and F) and sGC (Fig. 2G) were localized to the
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cytoplasm of luteal vascular cells. Labeling index, and expression of CD31 and eNOS were
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greater (P < 0.001) at early- than mid-luteal phase, and plane of nutrition did not affect any of
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these measurements; therefore, combined data are presented (Fig. 3). Expression of sGC protein
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was similar at early- and mid-luteal phases of the estrous cycle, and was not affected by
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nutritional plane (Fig. 3).
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Comparison of results obtained in this study with results generated from non-
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superovulated sheep (published recently by Bass et al. [1]) demonstrated that at mid-luteal, but
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not early-luteal phase, CL weights were less in FSH-treated than non-treated ewes. In addition, at
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early- and mid-luteal phases, cell proliferation and serum P4 were greater (P < 0.001), but
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vascularity (marked by CD31, eNOS and sGC expression) was less (P < 0.01) in superovulated
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than non-superovulated sheep (Fig. 4). The concentration of P4 in luteal tissues at early- and
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mid-luteal phases was similar in superovulated and non-superovulated ewes (Fig. 4). The number
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of CL was 5-6 fold greater (P < 0.001) in superovulated than non-superovulated C, O, and U
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sheep (Fig. 5).
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4. Discussion
The present experiment demonstrated that diet affected the number of ovulations, but did
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not affect several measurements of luteal function in FSH-treated sheep. However, CL weight,
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cell proliferation, vascularity, expression of eNOS protein, and serum P4 concentrations were
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affected by the phase of the estrous cycle in superovulated ewes. In addition, sGC protein
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expression and luteal P4 concentration were not affected by the stage of the estrous cycle or diet
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in superovulated ewes.
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In the present study, the mean number of CL/ewe varied from 9 to 14 depending on the nutritional plane, with the average being 13 CL/ewe in the C group for animals that responded to
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FSH treatment (e.g., CL number ≥4). Similarly, in our numerous previous studies, the number of
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CL in superovulated ewes fed a maintenance diet varied from 12 to 20 [25]. In superovulated
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cows, the number of CLs varied from 3-36 [37, 45-47]. In this study, the number of CL in
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superovulated U was less than in C or O ewes, and was similar to non-superovulated ewes [1].
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Furthermore, FSH-treatment did not increase ovulation rates for ~20% of sheep fed C diet, which
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is similar to data reported by others [25,48]. Thus, in addition to the well known phenomena that
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inadequate diet affects ovulation rates in non-treated ewes [1], this study demonstrated that diet
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also affected ovulation rates in superovulated sheep. The mechanisms of nutritional effects on
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ovulation and FSH-responses remain to be elucidated.
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In cows, the diverse effects of nutritional plane on superovulatory responses have been reported [45-47]. For FSH-treated cows fed 2x maintenance, the superovulatory response
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reflected by the number of CL was greater than in controls fed the maintenance diet [45].
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However, in FSH-treated cows fed 1.7x maintenance, the number of CL was less than in cows
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fed 0.7x maintenance [46]. In addition, the number of recovered embryos was less in FSH-
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treated cows with greater BCS (3.25 and 3.5) compared to lower BCS (2.75 and 3) indicating a
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greater superovulatory response in cows with lower BCS; however, these authors did not report
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the number of CL [49]. Furthermore, in PMSG-treated zebu cows, the number of CL was less in
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a “high” compared a “good” nutrition group [47]. Thus, effects of diet on superovulatory
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responses likely depend on hormonal-treatment protocol, species, and nutritional plane (e.g.,
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level of energy and composition of the diet and length of the nutritional treatment).
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Similar to previous reports, in this study, Ki67 was immunolocalized to the cell nuclei, but CD31, eNOS and sGC proteins were detected in the vascular cells of the ovine CL
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demonstrating a similar pattern of protein distribution in superovulated and non-superovulated
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ewes [1,12,50]. Others have demonstrated that luteal morphology, responsiveness of luteal cells
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to LH and dbcAMP, and the ratio of small to large luteal cells were similar in superovulated and
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non-superovulated ewes [39,41]. For superovulated ewes in this study, from the early- to mid-
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luteal phase of the estrous cycle, the pattern of changes of several measurements of luteal
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functions including the CL weight, cell proliferation, vascularity, and serum P4 concentration,
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were similar to those observed in non-superovulated ewes [1-3]. In comparison, greater
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proliferation rates and vascularity at early- than mid-luteal phases have been reported for several
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species including sheep, cows and primates [1,2,51-54]. Expression of eNOS in this study was
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greater in the early- than mid-luteal phase, which is similar to data reported previoulsy for non-
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superovulated ewes [12]. However, Bass et al. [1] observed that eNOS protein expression was
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greater at mid- than early-luteal phase of the estrous cycle. These differences in the
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determination of eNOS expression are likely due to the animal treatment and staining protocol.
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On the other hand, the pattern of sGC protein expression was similar in both superovulated and
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non-superovulated ewes [1]. Thus, these results indicate several similarities in luteal functions in
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superovulated and non-superovulated models.
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Comparison of selected measurements of luteal function in superovulated vs non-
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superovulated ewes demonstrated differences in cell proliferation, vascularity, expression of
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eNOS and sGC, and serum P4 concentration, in this study. In fact, the major differences between
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superovulated vs. non-superovulated ewes were in the serum P4 concentration, which has also
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been reported for several other species treated with FSH, including sheep, cows, rats, and
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humans [2,11,55-58]. Greater P4 concentrations in superovulated animals is due to the enhanced
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number of CL which consequently produce more P4 [25]. Furthermore, the luteal P4
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concentration was similar in superovulated vs. non-superovulated ewes indicating that the
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production of P4/luteal tissue unit is similar. This suggests that the steroidogenic process and its
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regulation may be similar in superovulated and non-superovulated ewes; however, future studies
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should compare the activity and expression of enzymes regulating luteal steroidogenesis in these
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two models.
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Interestingly, enhanced cell proliferation in the CL of superovulated ewes was not reflected by heavier CLs in this study. Although the weight of the CLs at the early luteal phase
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was similar, CL weight at the mid-luteal phase was less in superovulated than in non-
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superovulated ewes in this study. A similar observation concerning CL weight in superovulated
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and non-superovulated ewes was reported previously [25]. Thus, it seems that regulatory
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mechanisms exist which limit CL growth even at a high rate of cell proliferation. A high cell
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proliferation rate that is not reflected by changes in organ weight may be due to a smaller cell
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size; however, this hypothesis requires further investigation. Furthermore, vascularity (marked
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by expression of CD31, eNOS and sGC proteins) was less in superovulated than non-
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superovulated ewes [1]. However, reduced vascularity did not negatively affect P4 production, as
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measured by luteal P4 concentration.
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Our present study has demonstrated that luteal vascularity measured by expression of
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CD31, eNOS and sGC proteins was less in superovulated compared to non-superovulated ewes.
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However, in other ruminants, superovulatory treatments enhanced luteal vascularity and
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expression of genes involved in the regulation of blood vessel functions and angiogenesis
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[34,35]. In cows, enhanced vascular volume density, greater capillary diameter, and increased
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number of angiogenic structures in the CL at the early luteal phase of the estrous cycle was
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observed after eCG treatment, indicating that eCG has some angiogenic effects [35]. In
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buffaloes, mRNA and/or protein expression of FGF2, VEGF and their receptors was enhanced in
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luteal tissues at the early luteal phase of the estrous cycle in FSH-treated, superovulated
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compared to non-superovulated animals [34]. These differences in measurements of luteal
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vascular function are likely due to the species and superovulation protocols.
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In this study, diet did not affect any measurements of luteal functions. In our previous
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study, diet also did not affect serum and luteal P4 concentration in non-superovulated sheep [1].
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However, vascularity measured by CD31 expression was reduced at the early luteal phase in O
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and U ewes, and mid-luteal phase in O ewes, and cell proliferation was enhanced in O ewes at
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the early luteal phase compared to the C group in non-superovulated sheep [1]. These differences
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are likely due to the effects of FSH-treatment on luteal functions. Currently, the mechanism(s) of
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FSH-treatment effects on luteal functions is unknown, and this subject requires further study. In summary, this study demonstrated that diet affected ovulation rates in superovulated
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ewes, but not other measurements of luteal function. From the early- to mid-luteal phase, the
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pattern of changes in cell proliferation, vascularity, expression of eNOS and sGC, and serum P4
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concentration is similar in superovulated and non-superovulated ewes. However, CL weight at
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the mid-luteal phase, the rates of cell proliferation, vascularity, and expression of eNOS and sGC
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proteins, and serum P4 concentration at early- and mid-luteal phases differ in superovulated vs.
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non-superovulated ewes. Furthermore, the luteal P4 concentration is similar in superovulated and
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non-superovulated ewes, indicating that luteal cells produce P4 at similar rates in both models.
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Although the primary function of the CL, which is the rate of P4 production/tissue unit, is not
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affected by FSH treatment, and cells from superovulated animals respond to LH-treatment
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[41,59-62], superovulation models for studying luteal or other reproductive functions should be
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used with caution since several differences between these models exist.
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Acknowledgments
This project was supported by the USDA-AFRI grant 2011-67016-30174, and Hatch
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Projects ND01754 and ND01748 to ATGB and DAR. Aree Kraisoon, a Ph.D. candidate, was
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supported mainly by a grant from Thailand Research Fund (TRF) under Research and
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Researchers for Industries (RRI) and National Research Council of Thailand (NRCT).
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Figure legends
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Fig. 1. Average number of CL/ewe in control (C), overfed (O), or underfed (U) ewes.
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P<
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Fig. 2. Representative images of immunofluorescent staining of Ki67 (yellow; A and B), CD31
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(red; C and D), eNOS (red; E and F) and sGC (green; G) in luteal tissues from the early-
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(A, C, E and G) and mid- (B, D and F) luteal phases of the estrous cycle, respectively. Blue color in each image indicates DAPI nuclei staining. (H) Control staining where
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primary antibody was omitted. Note expression of CD31, eNOS, and sGC in blood
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vessels. Size bar for all images = 50 µm.
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Fig. 3. Expression of Ki67, CD31, eNOS and sGC in the CL from early- and mid- luteal phases of the estrous cycle. a,bP < 0.001, means ± SEM with different superscripts differ at the
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early- vs. mid-luteal phase within a specific measurement.
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Fig. 4. The effects of FSH-treatment on CL weight, Ki67, CD31, eNOS and sGC expression, and luteal and serum P4 concentration compared to non-treated ewes (arbitrary set as 1 and
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marked with black line) [1] at early- and mid-luteal phases of the estrous cycle; *P <
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0.01, compared to results generated from non-superovulated sheep.
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Fig. 5. The effects of FSH-treatment on the number of CL in control (C), overfed (O), and underfed (U) ewes compared to non-treated ewes (arbitrarily set as 1 and marked with
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black line) [1]. *P < 0.001, compared to results generated from non-superovulated sheep.
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Table 1. Tissue fixation, tissue section thickness, and antibodies used for immunohistochemistry to detect CD31, Ki67, eNOS, sGC, and FSHR proteins in the CL.
Ki67
Endothelial cell
NBF
Cell NBF proliferation
Endothelial nitric oxide synthase
Carnoy's
sGC
Soluble guanylate cyclase
NBF
5 µm
Ki67 mouse monoclonal, item # VP-k452, Vector Labs, Burlingame, CA; 1:100; overnight
Anti-eNOS/NOS III purified mouse, item # 610297, BD Biosciences, San Jose, CA; 1:250; overnight Guanylate cyclase β rabbit polyclonal, item # 160897, Cayman Chemical, Ann Arbor, MI; 1:500; 1 h
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eNOS
5 µm
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CD31
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5 µm
2⁰antibody source, dilution and incubation time CF 633 goat antirabbit, item # 20122-0.5mL, Biotium, San Francisco, CA; 1:100; 1 h Alexa 568 goat anti-mouse, item # A110040, Life Technologies, Carlsbad, CA; 1:200; 1 h Alexa 647 goat anti-mouse, item # A21235, Invitrogen, Eugene, OR; 1:200; 1 h CF 633 goat antirabbit, item # 20122-0.5mL, Biotium, San Francisco, CA; 1:100; 1 h
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1⁰ antibody source, Tissue dilution and thickness incubation time CD31 rabbit polyclonal, item # ab28364, Abcam 15 µm Biotech Company, San Francisco, CA; 1:100; overnight
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Reference
[1]
[1]
[1]
[1]
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Table 2. Weight of CL, and serum and luteal P4 concentrations at the early- and mid-luteal phases of the estrous cycle.
Early-luteal
Mid-luteal
CL weight (mg)
210.0 ± 17.0
336.0±22.0
P < 0.02
Serum P4 concentration (ng/ml)
8.0 ± 1.0
10.3 ± 1.3
P < 0.09
Luteal P4 concentration (µg/g of tissue)
8.3 ± 0.8
SC 9.1 ± 0.8
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P-value
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Measurement
P > 0.1
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3 0 O Nutritional plan
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Fig. 3. 24
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b 4
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CD31
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eNOS
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sGC
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Fig. 4.
Early
Mid
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0 Ki67
CD31
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eNOS
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Fold change (compared to not FSH-treated)
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serum P4
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Cell proliferation, vascularity and expression of endothelial nitric oxide synthase depends on the stage of luteal development but not diet in superovulated ewes Serum progesterone concentration is greater in superovulated than non-superovulated ewes Luteal progesterone concentration is similar in superovulated than non-superovulated ewes FSH-induced superovulation alters selected luteal functions in sheep.
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S0739-7240(17)30108-X
DOI:
10.1016/j.domaniend.2017.08.002
Reference:
DAE 6277
To appear in:
Domestic Animal Endocrinology
Received Date: 16 May 2017 Revised Date:
28 July 2017
Accepted Date: 5 August 2017
Please cite this article as: Kraisoon A, Redmer DA, Bass CS, Navanukraw C, Dorsam ST, Valkov V, Reyaz A, Grazul-Bilska AT, Corpora lutea in superovulated ewes fed different planes of nutrition, Domestic Animal Endocrinology (2017), doi: 10.1016/j.domaniend.2017.08.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Corpora lutea in superovulated ewes fed different planes of nutrition*
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A. Kraisoona, D.A. Redmerb, C.S. Bassb, C. Navanukrawac, S.T. Dorsamb, V. Valkovb,
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A. Reyazb, and A.T. Grazul-Bilskab**
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a
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Thailand;
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b
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c
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Department of Animal Sciences, North Dakota State University, Fargo, ND, USA
Agricultural Biotechnology Research Center for Sustainable Economy (ABRCSE), Khon Kaen
University, Khon Kaen, Thailand.
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Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen,
*A preliminary report of these data was presented at the Western Section of the American
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Society of Animal Sciences meetings in June 2017.
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**Corresponding author. Tel.: +1 701 231 7992; fax: +1 701 231 7590.
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E-mail address: [email protected] (A.T. Grazul-Bilska).
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ABSTRACT The corpus luteum (CL) is an ovarian structure which is critical for the maintenance of reproductive cyclicity and pregnancy support. Diet and/or diet components may affect some
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luteal functions. FSH is widely used to induce multiple follicle development and superovulation.
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We hypothesized that FSH would affect luteal function in ewes fed different nutritional planes.
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Therefore, the aim of this study was to determine if FSH-treatment affects 1) ovulation rate; 2)
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CL weight; 3) cell proliferation; 4) vascularity; 5) expression of endothelial nitric oxide (eNOS)
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and soluble guanylyl cyclase (sGC) proteins; and 6) luteal and serum progesterone (P4)
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concentration in control (C), overfed (O) and underfed (U) ewes at the early- and mid-luteal
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phases. In addition, data generated from this study were compared to data obtained from non-
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superovulated sheep and described by Bass et al. [1]. Ewes were categorized by weight, and
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randomly assigned into nutrition groups: C (2.14 Mcal/kg; n=11), O (2xC; n=12), and U (0.6xC;
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n=11). Nutritional treatment was initiated 60 d prior to d 0 of the estrous cycle. Ewes were
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injected with FSH on d 13-15 of the first estrous cycle, and blood samples and ovaries were
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collected at early- and mid-luteal phases of the second estrous cycle. The number of CL/ewe was
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determined, and CL were dissected and weighed. CL were fixed for evaluation of expression of
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Ki67 (a proliferating cell marker), CD31 (an endothelial cell marker), and eNOS and sGC
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proteins using immunohistochemistry and image analysis. From d 0 until tissue collection, C
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maintained, O gained and U lost BW. The CL number was greater (P < 0.03) in C and O than U.
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Weights of CL, cell proliferation, vascularity, and eNOS but not sGC expression were greater (P
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< 0.001), and serum, but not luteal tissue, P4 concentrations tended to be greater (P = 0.09) at the
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early- than mid-luteal phase. Comparisons of CL measurements demonstrated greater (P < 0.01)
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cell proliferation and serum P4 concentration, but less vascularity at the early and mid-luteal
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phases, and less CL weight at the mid-luteal phase in superovulated than non-superovulated
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ewes; however, concentration of P4 in luteal tissues was similar in both groups. Thus, in
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superovulated ewes, luteal cell proliferation and vascularity, expression of eNOS, and serum P4
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concentration depends on the stage of luteal development, but not diet. Comparison to control
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ewes demonstrated several differences and some similarities in luteal functions after FSH-
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induced superovulation.
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Keywords: Corpora lutea; eNOS; vascularity; FSH; superovulation; sheep
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1. Introduction
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The mammalian corpus luteum (CL), the major source of progesterone (P4) in females, is a transient endocrine gland, which grows, differentiates and regresses during each estrous cycle
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and pregnancy, and plays a major role in reproductive processes [2-7]. Growth, differentiation
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and regression of the CL depend on a balance between luteotropic (e.g., luteinizing hormone)
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and luteolytic (e.g., prostaglandin F2α) factors that subsequently regulate P4 secretion [6,7]. In
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addition, luteal functions may be affected by selected environmental factors, such as nutrition
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[1,8-9]. For example, overfeeding resulted in enhanced serum P4 concentration, and inadequate
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diet (e.g., excess or restriction) altered luteal vascularity, cell proliferation, and mRNA
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expression of selected angiogenic factors in non-pregnant sheep [1,8].
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Dynamic changes within the CL during each estrous cycle include cell proliferation, and
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the establishment and growth of the vascular bed during early- and mid-luteal phases of the
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estrous cycle [1]. The CL consists of several cell types including parenchymal steroidogenic
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small and large luteal cells, vascular cells (endothelial cells, pericytes and smooth muscle cells),
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fibroblasts, immune cells and others [6,7]. It has been suggested that these cell types interact to
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maintain normal luteal function [3,7]. The CL is highly vascularized, and vascular cells comprise
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more than 50% of the total cells in the CL [4]. Vascular growth and function is regulated by
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several angiogenic and growth factors including those associated with production of nitric oxide
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(NO) [1,4,5]. Nitric oxide is a free radical gaseous molecule that is produced in endothelial cells
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by endothelial NO synthase (eNOS)-mediated breakdown of L-arginine [11]. In addition, NO
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diffuses rapidly from endothelial cells into the underlying smooth muscle cells or pericytes
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where it activates soluble guanylate cyclase (sGC). Both, eNOS and sGC are expressed in CL of
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non-pregnant sheep [1,12,13]. Furthermore, the NO system is involved in the regulation of luteal
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functions such as steroid production, angiogenesis, vascularity, apoptosis and luteolysis in
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several species [14-24].
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Follicle stimulating hormone has been widely used to stimulate multiple follicle development and/or superovulation in mammalian species [25-30]. Superovulated models are
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predominantly used for embryo production and to study selected reproductive processes
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including luteal functions in several species [24,31-37]. In sheep and cows, superovulation has
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resulted in greater number of CL and serum P4 concentrations compared to non-treated animals
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[25,27,37-40]. Furthermore, morphology of the CL, and response to LH was similar for
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superovulated and non-superovulated ewes [39,41], but in superovulated cows, expression of
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several genes in luteal tissues was altered, and vascular volume density in CL was increased
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[34,35,37]. However, only very few studies (cited above) evaluated luteal functions in
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superovulated animals. Since superovulation protocols are being used in animal production and
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research, and human medicine [25-30,38-41], and the CL is critical for maintaining normal
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reproductive processes and thus, fertility [6,7], study of luteal functions in superovulated models
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are warranted. Therefore, the aim of this study was to characterize the CL from superovulated
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ewes fed control, excess and restricted diets by determining cell proliferation, vascularity
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(marked by CD31 expression), expression of eNOS and sGC proteins, and serum and luteal P4
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concentrations at the early- and mid-luteal stages of the estrous cycle. These stages correspond to
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the rapid growth and differentiating phases of luteal development [2,3]. Furthermore, luteal
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measurements generated from this study were compared to the same measurements generated
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from non-superovulated ewes in our laboratory [1].
2. Materials and Methods
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2.1. Animals and Experimental Design
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All animal procedures performed were approved by the North Dakota State University (NDSU) Institutional Animal Care and Use Committee (#A12013). The study was initiated
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during the normal breeding season in August and finished in December.
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Non-pregnant, non-lactating Rambouillet ewes between 3 to 5 years of age and of similar
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genetic background were individually penned at the Animal Nutrition and Physiology Center on
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the NDSU campus. Ewes were stratified by body weight (BW) and randomly assigned into one
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of three dietary groups: maintenance-control (C; 100% National Research Council [NRC]
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requirements; 2.4 Mcal of metabolizable energy [ME]/kg BW), overfed (O; 200% NRC
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requirements), or underfed (U; 60% NRC requirements), as previously described [1,8]. Diets
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were initiated 60 d prior to the onset of estrus (d 0). Ewes were fed their individual diets twice
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daily at 0800 and 1500h for the duration of the experiment, and ewes were weighed once weekly.
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Individual rations were adjusted weekly to ensure the proper BW (e.g., C, O and U) was
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achieved at d 0, and maintained throughout the estrous cycle and until completion of the
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experiment. Estrus was synchronized by insertion of a controlled internal drug release (CIDR)
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device for 14 d. Approximately 36 h after removal of the CIDR, ewes were in estrus, which was
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treated as d 0 of the estrous cycle [1,42,43]. Ewes (n = 34) were injected twice daily (morning and evening) with follicle stimulating
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hormone (FSH-P; Sioux Biochemical, Sioux Center, IA, USA) on d 13 to 15 of the first estrous
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cycle (5 mg/injection, 4 mg/injection, or 3 mg/injection, respectively) [43]. This protocol of
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FSH-treatment, reported extensively by our group, has caused multiple ovulations resulting in
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16±0.5 CL per sheep in years 1989-2005 [25]. Initial BW and BCS were similar for all groups
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(55.9 ± 1.5 kg and 2.9 ± 0.1, respectively). Throughout the study, C (n = 11) maintained BW, O
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(n = 11) ewes gained (P < 0.001) 13.1 ± 1 kg and BCS increased (P < 0.01) by 1.0 ± 0.1, and U
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(n = 12) ewes lost (P < 0.001) 7.7 ± 1 kg and BCS decreased (P < 0.01) by 0.7 ± 0.1, similar to
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our previous studies [1,43,44].
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2.2. Tissue and Blood Collection, Immunohistochemistry and Image Analysis Ovaries and blood samples were collected at early- (d 5) and mid- (d 10) luteal phases of the second estrous cycle [43]. Blood samples were centrifuged (20 min at 1,500 g), and serum
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stored at -20˚C until P4 analysis. Number of CL was recorded, and CLs were dissected from
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ovaries and weighed. Randomly selected CL from each ewe was divided into three portions: the
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first portion was fixed in 10% neutral buffered formalin (NBF) for immunohistochemistry, the
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second portion was fixed in Carnoy’s solution for immunohistochemistry, and the third portion
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(approximately 20-50 mg) was immediately frozen on dry ice and stored at -80 ˚C until
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homogenization in PBS (100 mg/1 ml) for evaluation of P4 concentrations.
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Immunohistochemistry was performed as described in detail by Bass et al. [1]. Tissue sections underwent antigen recovery (2100 Retriever, Prestige Medical, Lancashire, England) for
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20 minutes in a 10 mM sodium citrate buffer with 0.05% Tween (pH 6), were washed and
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blocked with 10% normal goat serum for 20 minutes at room temperature, incubated with a
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specific primary antibody, and then with a secondary antibody. Table 1 presents fixatives used,
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tissue thickness, source and dilution of primary and secondary antibodies for all antigens
136
immunodetected. Tissue sections stained for CD31, Ki67, eNOS and sGC were cover slipped
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using Prolong Gold with DAPI mounting media (Life Technologies, Eugene, OR, USA).
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Photomicrographs were taken with a Zeiss Imager M2 epifluorescence microscope
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equipped with an AxioCam HRm camera (Zeiss Inc., Thornwood, NY, USA). Images used for
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image analysis were taken from areas where distribution of steroidogenic and accessory cells
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was uniform; areas with connective tissue were avoided. The percentage of the area that
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exhibited positive staining for CD31, eNOS or sGC was evaluated quantitatively with an image
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analysis software package (Image Pro-Plus, Media Cybernetics, Silver Spring, MD) as described
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previously [1]. Labeling index (LI) was calculated as the percentage (%) of proliferating Ki67-
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positive cells out of the total number of cells (DAPI nuclear staining) within the tissue area. For
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each CL, four randomly chosen fields were evaluated in each tissue section. Background
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fluorescence was minimal and was adjusted to the same level for each section by the image
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analysis system. Data are expressed as the percentage of positive staining out of total tissue area
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within each field.
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2.3. Progesterone Analysis
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Progesterone concentration in serum and luteal tissues was determined using a solid phase chemiluminescence, competitive binding immunoassay (Immulite 1000, Siemens, PA,
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USA), as previously described [1,8]. Each sample was run in duplicate. Intra-assay CVs were
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6.1% for serum and 5.5% for luteal P4.
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2.4. Statistical analysis
Data were analyzed statistically using the GLM procedure of SAS 9.2 (Cary, NC, USA).
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The model included plane of nutrition, phase of the estrous cycle, and their interactions. For
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comparison between non-superovulated vs. superovulated ewes, the model included FSH- vs.
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saline-treatment, and interactions. Data presented as percentage were analyzed using Chi square.
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When the F-test was significant (P ≤ 0.05), the differences between specific means were
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separated by using the least significant difference. Data are expressed as mean ± SEM.
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3. Results
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The weights of CL and serum P4 concentrations were greater (P < 0.02-0.09) at midthan early-luteal phase, and were not affected by nutritional plane; therefore, combined data for
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the three nutritional groups are presented in Table 2. The concentrations of P4 in luteal tissues
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were similar (P > 0.1) at early- and mid -luteal phases of the estrous cycle (Table 2). The number
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of CL, and thus ovulation rate, was greater (P < 0.03) in O and C when compared to U (Fig. 1).
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The number of CL ranged from 4-29, 4-27 and 4-22 in C, O and U, respectively. The proportion
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of ewes that did not respond to FSH treatment (the number of CL ≤ 3) was 19%, and it was not
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affected by the plane of nutrition (21, 7 and 27% in C, O and U, respectively).
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Ki67, CD31, eNOS and sGC proteins were immunodetected in CL from early- and midluteal phases of the estrous cycle (Fig. 2 and 3). Ki67 (Fig. 2A and B) was localized to cell
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nuclei, but CD31 (Fig. 2C and D), eNOS (Fig. 2E and F) and sGC (Fig. 2G) were localized to the
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cytoplasm of luteal vascular cells. Labeling index, and expression of CD31 and eNOS were
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greater (P < 0.001) at early- than mid-luteal phase, and plane of nutrition did not affect any of
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these measurements; therefore, combined data are presented (Fig. 3). Expression of sGC protein
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was similar at early- and mid-luteal phases of the estrous cycle, and was not affected by
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nutritional plane (Fig. 3).
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Comparison of results obtained in this study with results generated from non-
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superovulated sheep (published recently by Bass et al. [1]) demonstrated that at mid-luteal, but
184
not early-luteal phase, CL weights were less in FSH-treated than non-treated ewes. In addition, at
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early- and mid-luteal phases, cell proliferation and serum P4 were greater (P < 0.001), but
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vascularity (marked by CD31, eNOS and sGC expression) was less (P < 0.01) in superovulated
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than non-superovulated sheep (Fig. 4). The concentration of P4 in luteal tissues at early- and
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mid-luteal phases was similar in superovulated and non-superovulated ewes (Fig. 4). The number
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of CL was 5-6 fold greater (P < 0.001) in superovulated than non-superovulated C, O, and U
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sheep (Fig. 5).
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4. Discussion
The present experiment demonstrated that diet affected the number of ovulations, but did
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not affect several measurements of luteal function in FSH-treated sheep. However, CL weight,
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cell proliferation, vascularity, expression of eNOS protein, and serum P4 concentrations were
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affected by the phase of the estrous cycle in superovulated ewes. In addition, sGC protein
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expression and luteal P4 concentration were not affected by the stage of the estrous cycle or diet
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in superovulated ewes.
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In the present study, the mean number of CL/ewe varied from 9 to 14 depending on the nutritional plane, with the average being 13 CL/ewe in the C group for animals that responded to
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FSH treatment (e.g., CL number ≥4). Similarly, in our numerous previous studies, the number of
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CL in superovulated ewes fed a maintenance diet varied from 12 to 20 [25]. In superovulated
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cows, the number of CLs varied from 3-36 [37, 45-47]. In this study, the number of CL in
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superovulated U was less than in C or O ewes, and was similar to non-superovulated ewes [1].
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Furthermore, FSH-treatment did not increase ovulation rates for ~20% of sheep fed C diet, which
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is similar to data reported by others [25,48]. Thus, in addition to the well known phenomena that
207
inadequate diet affects ovulation rates in non-treated ewes [1], this study demonstrated that diet
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also affected ovulation rates in superovulated sheep. The mechanisms of nutritional effects on
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ovulation and FSH-responses remain to be elucidated.
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In cows, the diverse effects of nutritional plane on superovulatory responses have been reported [45-47]. For FSH-treated cows fed 2x maintenance, the superovulatory response
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reflected by the number of CL was greater than in controls fed the maintenance diet [45].
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However, in FSH-treated cows fed 1.7x maintenance, the number of CL was less than in cows
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fed 0.7x maintenance [46]. In addition, the number of recovered embryos was less in FSH-
215
treated cows with greater BCS (3.25 and 3.5) compared to lower BCS (2.75 and 3) indicating a
216
greater superovulatory response in cows with lower BCS; however, these authors did not report
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the number of CL [49]. Furthermore, in PMSG-treated zebu cows, the number of CL was less in
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a “high” compared a “good” nutrition group [47]. Thus, effects of diet on superovulatory
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responses likely depend on hormonal-treatment protocol, species, and nutritional plane (e.g.,
220
level of energy and composition of the diet and length of the nutritional treatment).
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Similar to previous reports, in this study, Ki67 was immunolocalized to the cell nuclei, but CD31, eNOS and sGC proteins were detected in the vascular cells of the ovine CL
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demonstrating a similar pattern of protein distribution in superovulated and non-superovulated
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ewes [1,12,50]. Others have demonstrated that luteal morphology, responsiveness of luteal cells
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to LH and dbcAMP, and the ratio of small to large luteal cells were similar in superovulated and
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non-superovulated ewes [39,41]. For superovulated ewes in this study, from the early- to mid-
227
luteal phase of the estrous cycle, the pattern of changes of several measurements of luteal
228
functions including the CL weight, cell proliferation, vascularity, and serum P4 concentration,
229
were similar to those observed in non-superovulated ewes [1-3]. In comparison, greater
230
proliferation rates and vascularity at early- than mid-luteal phases have been reported for several
231
species including sheep, cows and primates [1,2,51-54]. Expression of eNOS in this study was
232
greater in the early- than mid-luteal phase, which is similar to data reported previoulsy for non-
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superovulated ewes [12]. However, Bass et al. [1] observed that eNOS protein expression was
234
greater at mid- than early-luteal phase of the estrous cycle. These differences in the
235
determination of eNOS expression are likely due to the animal treatment and staining protocol.
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On the other hand, the pattern of sGC protein expression was similar in both superovulated and
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non-superovulated ewes [1]. Thus, these results indicate several similarities in luteal functions in
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superovulated and non-superovulated models.
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Comparison of selected measurements of luteal function in superovulated vs non-
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superovulated ewes demonstrated differences in cell proliferation, vascularity, expression of
241
eNOS and sGC, and serum P4 concentration, in this study. In fact, the major differences between
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superovulated vs. non-superovulated ewes were in the serum P4 concentration, which has also
243
been reported for several other species treated with FSH, including sheep, cows, rats, and
244
humans [2,11,55-58]. Greater P4 concentrations in superovulated animals is due to the enhanced
245
number of CL which consequently produce more P4 [25]. Furthermore, the luteal P4
246
concentration was similar in superovulated vs. non-superovulated ewes indicating that the
247
production of P4/luteal tissue unit is similar. This suggests that the steroidogenic process and its
248
regulation may be similar in superovulated and non-superovulated ewes; however, future studies
249
should compare the activity and expression of enzymes regulating luteal steroidogenesis in these
250
two models.
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Interestingly, enhanced cell proliferation in the CL of superovulated ewes was not reflected by heavier CLs in this study. Although the weight of the CLs at the early luteal phase
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was similar, CL weight at the mid-luteal phase was less in superovulated than in non-
254
superovulated ewes in this study. A similar observation concerning CL weight in superovulated
255
and non-superovulated ewes was reported previously [25]. Thus, it seems that regulatory
256
mechanisms exist which limit CL growth even at a high rate of cell proliferation. A high cell
257
proliferation rate that is not reflected by changes in organ weight may be due to a smaller cell
258
size; however, this hypothesis requires further investigation. Furthermore, vascularity (marked
259
by expression of CD31, eNOS and sGC proteins) was less in superovulated than non-
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superovulated ewes [1]. However, reduced vascularity did not negatively affect P4 production, as
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measured by luteal P4 concentration.
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Our present study has demonstrated that luteal vascularity measured by expression of
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CD31, eNOS and sGC proteins was less in superovulated compared to non-superovulated ewes.
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However, in other ruminants, superovulatory treatments enhanced luteal vascularity and
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expression of genes involved in the regulation of blood vessel functions and angiogenesis
266
[34,35]. In cows, enhanced vascular volume density, greater capillary diameter, and increased
267
number of angiogenic structures in the CL at the early luteal phase of the estrous cycle was
268
observed after eCG treatment, indicating that eCG has some angiogenic effects [35]. In
269
buffaloes, mRNA and/or protein expression of FGF2, VEGF and their receptors was enhanced in
270
luteal tissues at the early luteal phase of the estrous cycle in FSH-treated, superovulated
271
compared to non-superovulated animals [34]. These differences in measurements of luteal
272
vascular function are likely due to the species and superovulation protocols.
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In this study, diet did not affect any measurements of luteal functions. In our previous
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study, diet also did not affect serum and luteal P4 concentration in non-superovulated sheep [1].
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However, vascularity measured by CD31 expression was reduced at the early luteal phase in O
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and U ewes, and mid-luteal phase in O ewes, and cell proliferation was enhanced in O ewes at
277
the early luteal phase compared to the C group in non-superovulated sheep [1]. These differences
278
are likely due to the effects of FSH-treatment on luteal functions. Currently, the mechanism(s) of
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FSH-treatment effects on luteal functions is unknown, and this subject requires further study. In summary, this study demonstrated that diet affected ovulation rates in superovulated
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ewes, but not other measurements of luteal function. From the early- to mid-luteal phase, the
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pattern of changes in cell proliferation, vascularity, expression of eNOS and sGC, and serum P4
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concentration is similar in superovulated and non-superovulated ewes. However, CL weight at
284
the mid-luteal phase, the rates of cell proliferation, vascularity, and expression of eNOS and sGC
285
proteins, and serum P4 concentration at early- and mid-luteal phases differ in superovulated vs.
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non-superovulated ewes. Furthermore, the luteal P4 concentration is similar in superovulated and
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non-superovulated ewes, indicating that luteal cells produce P4 at similar rates in both models.
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Although the primary function of the CL, which is the rate of P4 production/tissue unit, is not
289
affected by FSH treatment, and cells from superovulated animals respond to LH-treatment
290
[41,59-62], superovulation models for studying luteal or other reproductive functions should be
291
used with caution since several differences between these models exist.
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Acknowledgments
This project was supported by the USDA-AFRI grant 2011-67016-30174, and Hatch
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Projects ND01754 and ND01748 to ATGB and DAR. Aree Kraisoon, a Ph.D. candidate, was
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supported mainly by a grant from Thailand Research Fund (TRF) under Research and
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Researchers for Industries (RRI) and National Research Council of Thailand (NRCT).
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Figure legends
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Fig. 1. Average number of CL/ewe in control (C), overfed (O), or underfed (U) ewes.
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P<
0.03, means ±SEM with different superscripts differ.
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Fig. 2. Representative images of immunofluorescent staining of Ki67 (yellow; A and B), CD31
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(red; C and D), eNOS (red; E and F) and sGC (green; G) in luteal tissues from the early-
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(A, C, E and G) and mid- (B, D and F) luteal phases of the estrous cycle, respectively. Blue color in each image indicates DAPI nuclei staining. (H) Control staining where
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primary antibody was omitted. Note expression of CD31, eNOS, and sGC in blood
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vessels. Size bar for all images = 50 µm.
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Fig. 3. Expression of Ki67, CD31, eNOS and sGC in the CL from early- and mid- luteal phases of the estrous cycle. a,bP < 0.001, means ± SEM with different superscripts differ at the
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early- vs. mid-luteal phase within a specific measurement.
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Fig. 4. The effects of FSH-treatment on CL weight, Ki67, CD31, eNOS and sGC expression, and luteal and serum P4 concentration compared to non-treated ewes (arbitrary set as 1 and
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marked with black line) [1] at early- and mid-luteal phases of the estrous cycle; *P <
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0.01, compared to results generated from non-superovulated sheep.
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Fig. 5. The effects of FSH-treatment on the number of CL in control (C), overfed (O), and underfed (U) ewes compared to non-treated ewes (arbitrarily set as 1 and marked with
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black line) [1]. *P < 0.001, compared to results generated from non-superovulated sheep.
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Table 1. Tissue fixation, tissue section thickness, and antibodies used for immunohistochemistry to detect CD31, Ki67, eNOS, sGC, and FSHR proteins in the CL.
Ki67
Endothelial cell
NBF
Cell NBF proliferation
Endothelial nitric oxide synthase
Carnoy's
sGC
Soluble guanylate cyclase
NBF
5 µm
Ki67 mouse monoclonal, item # VP-k452, Vector Labs, Burlingame, CA; 1:100; overnight
Anti-eNOS/NOS III purified mouse, item # 610297, BD Biosciences, San Jose, CA; 1:250; overnight Guanylate cyclase β rabbit polyclonal, item # 160897, Cayman Chemical, Ann Arbor, MI; 1:500; 1 h
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eNOS
5 µm
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CD31
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5 µm
2⁰antibody source, dilution and incubation time CF 633 goat antirabbit, item # 20122-0.5mL, Biotium, San Francisco, CA; 1:100; 1 h Alexa 568 goat anti-mouse, item # A110040, Life Technologies, Carlsbad, CA; 1:200; 1 h Alexa 647 goat anti-mouse, item # A21235, Invitrogen, Eugene, OR; 1:200; 1 h CF 633 goat antirabbit, item # 20122-0.5mL, Biotium, San Francisco, CA; 1:100; 1 h
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1⁰ antibody source, Tissue dilution and thickness incubation time CD31 rabbit polyclonal, item # ab28364, Abcam 15 µm Biotech Company, San Francisco, CA; 1:100; overnight
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Antigen Marker of
Reference
[1]
[1]
[1]
[1]
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Table 2. Weight of CL, and serum and luteal P4 concentrations at the early- and mid-luteal phases of the estrous cycle.
Early-luteal
Mid-luteal
CL weight (mg)
210.0 ± 17.0
336.0±22.0
P < 0.02
Serum P4 concentration (ng/ml)
8.0 ± 1.0
10.3 ± 1.3
P < 0.09
Luteal P4 concentration (µg/g of tissue)
8.3 ± 0.8
SC 9.1 ± 0.8
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P-value
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Measurement
P > 0.1
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3 0 O Nutritional plan
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eNOS
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Mid
*
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0 Ki67
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eNOS
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serum P4
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Cell proliferation, vascularity and expression of endothelial nitric oxide synthase depends on the stage of luteal development but not diet in superovulated ewes Serum progesterone concentration is greater in superovulated than non-superovulated ewes Luteal progesterone concentration is similar in superovulated than non-superovulated ewes FSH-induced superovulation alters selected luteal functions in sheep.
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