NIH Public Access Author Manuscript Anim Reprod Sci. Author manuscript; available in PMC 2015 August 01.
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Published in final edited form as: Anim Reprod Sci. 2014 August ; 148(0): 121–129. doi:10.1016/j.anireprosci.2014.05.002.
The effect of leptin on luteal angiogenic factors during the luteal phase of the estrous cycle in goats Jessica R. Wiles1, Robin A. Katchko1, Elizabeth A. Benavides1, Chad W. O’Gorman1, Jean M. Escudero2, Duane H. Keisler3, Randy L. Stanko1,4, and Michelle R. Garcia1,* 1Department
of Animal, Rangeland & Wildlife Science, Dick and Mary Lewis Kleberg College of Agriculture, Natural Resources and Human Sciences, Texas A&M University-Kingsville, Kingsville, TX 78363 2Department
of Biological and Health Sciences, College of Arts and Sciences, Texas A&M University-Kingsville, Kingsville, TX 78363
NIH-PA Author Manuscript
3Division
of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, MO 65211
4Animal
Reproduction Laboratory, Texas A&M University AgriLife Research Station, Beeville, TX
78102
Abstract
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Fibroblast growth factor 2 (FGF2), angiopoietin 1 (Ang1), and vascular endothelial growth factor (VEGF) are angiogenic factors implicated in the vascular development of the corpus luteum (CL). Each factor is regulated or influenced by leptin in non-ovarian tissues. Moreover, leptin and its receptor, ObRb, have been identified in luteal tissue throughout the luteal phase. Therefore, leptin is hypothesized to influence luteal vasculature through the regulation of FGF2, Ang1, and VEGF. Multiparous, cycling crossbred female goats (does) were allocated to early (n=12), mid (n=8), and late (n=11) stages of the luteal phase for CL collection. Luteal tissue was harvested and either snap frozen in liquid N2, paraffin embedded, or cultured with leptin (0, 10−12, 10−11, 10−10, 10−9, 10−8 M). Tissue was analyzed for FGF2, Ang1, VEGF, ObRb, and leptin expression. Angiopoietin 1, FGF2, VEGF expression was higher (P≤0.001) in the mid-luteal stage than the early stage. Expression decreased (P≤0.001) during the late luteal stage with the exception of VEGF, which remained elevated. In contrast, leptin and ObRb were lowest (P≤0.003) during the mid-luteal stage compared to the early and late stages. All factors were detected in and/or around vessels in early stage tissue compared to mid and late stages. Leptin stimulated (P≤0.02) Ang1, FGF2, and VEGF
*
Corresponding Author: Texas A&M University-Kingsville, Department of Animal, Rangeland & Wildlife Sciences, Kleberg Ag. Bldg. Rm 133, Kingsville, TX 78363, USA. Tel: +1-361-593-3197; fax: +1-361-593-3788; [email protected]. Competing interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Authors’ contributions JRW participated in study design, acquisition, analysis and interpretation of data, and in preparation of the manuscript. RAK, EAB, and CWO participated in acquisition and interpretation of the data. JME, DHK, and RLS participated in the analysis of data. As Principle Investigator, MRG participated in the intellectual and experimental design of the study, data acquisition, analysis and interpretation, as well as writing and revising the manuscript. All authors read and approved the final manuscript. Publisher's Disclaimer: 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 citable 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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expression only in early stage luteal cultures. Collectively, these data provide evidence that leptin may be involved in the luteal angiogenic process during the early stage of CL formation.
NIH-PA Author Manuscript
Keywords leptin corpus luteum angiogenesis goats
1. Introduction
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Abnormal vasculature of the CL is a defect that leads to abnormal development and decreased progesterone production (Glock and Brumsted, 1995; Hazzard and Stouffer, 2000). Identifying and understanding the underlying mechanisms of luteal angiogenesis may lead to a reduction in luteal-related infertility and alternate methodology for the regulation of reproductive cycles. In the ovary, angiogenesis is a cyclical, recurring process that takes place following ovulation. Upon ovulation, the basement membrane between the granulosa and thecal layers disassociates allowing the thecal capillaries to invade the avascular granulosa layer and antral space forming a nascent network of capillaries to nourish the developing CL. The luteal neo-vascularization process is attributed to the biological activity of FGF2, Ang1, and VEGF. Both FGF2 and VEGF promote capillary membrane destabilization, endothelial cell differentiation, proliferation, migration, and vascular tube formation in human, bovine, and ovine luteal tissue (Suzuki et al., 1998; Reynolds and Redmer, 1998). Maturation and stabilization of nascent vessels is then promoted by Ang1 through the recruitment of stromal support cells, including pericytes and smooth muscle cells (Shalaby et al., 1995). Interestingly, each of these angiogenic regulatory factors is influenced by the adipogenic hormone leptin (Aleffi et al., 2005, Cao et al., 2001).
NIH-PA Author Manuscript
Both leptin and its receptor (ObRb) have been identified in normal and polycystic ovaries of various species (Loffler et al., 2001; Ruiz-Cortez et al., 2000; Ryan et al., 2003; MunozGutierrez et al., 2005), particularly in steroidogenically active follicles and luteal tissue. This would infer that leptin may be involved in steroidogenic processes (Kendall et al., 2004). However, in order to substantially modify steroid production cells must be incubated with leptin in the presence of a growth promotant (Spicer and Francisco 1997; Spicer and Francisco 1998; Zachow and Magoffin 1997; Karlsson et al., 1997; Brannian et al., 1999), which suggests that an ancillary role for leptin in ovarian tissue, such as angiogenesis, may exist. In support, ObRb has been identified in ovarian vascular endothelial cells in rodents (Ryan et al., 2003) and its expression in the porcine CL is highest during luteal development, a period of intense angiogenic activity (Ruiz-Cortez et al., 2000). Furthermore, leptin alone is capable of stimulating angiogenesis in the cornea of the eye (Sierra-Honigmann et al., 1998) and upregulates the expression of angiogenic factors in nonovarian tissue (Aleffi et al., 2005, Cao et al., 2001). Collectively, the evidence supports the supposition that leptin may be involved in ovarian angiogenesis, specifically vascularization of a developing CL. Therefore, it is hypothesized that leptin will influence the expression of angiogenic growth factors, FGF2, Ang1, and VEGF, in developing luteal tissue. The objectives were to: 1.) characterize FGF2, Ang1, VEGF, leptin, and ObRb expression at different stages of the luteal phase of the estrous cycle, and 2.) determine the effect of leptin
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on the expression of FGF2, Ang1, and VEGF in dispersed luteal tissue during the early, mid, and late stages of the luteal phase. Understanding the mechanisms of the development of luteal tissue may lead to a future reduction in the occurrence of infertility associated with luteal defects.
2. Materials and methods Institutional Animal Care and Use Committee at Texas A&M University-Kingsville (TAMUK) approved the animal care procedures used for this study. 2.1. Animals
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Thirty-one mature, cycling crossbred (Boer × Spanish) female goats (does), Capra aegagrus hircus, averaging 11 months of age and weighing 38.2 ± 2.3 kg at date of tissue collection, from the Texas A&M University-Kingsville (TAMUK) farm were utilized. Animals were randomly allocated for CL tissue collection at either the early (n=12), mid (n=8), or late (n=11) stages of the luteal phase of the estrous cycle. Early, mid, and late stages of the luteal phase are defined by the day of the estrous cycle beginning with day of initial onset of estrus (D0). Therefore, ‘early stage’ CL were collected on D3 of the estrous cycle which is ~35-42 h post-ovulation, while ‘mid stage’ CL were collected on D10 of the estrous cycle, and ‘late stage’ CL were collected on D15 of the normally 21-day caprine estrous cycle. Tissue was collected to satisfy minimum requirements of 6 does/luteal stage, not exceeding 12 does/ luteal stage, for tissue characterization analysis and cell cultures. The specifically defined luteal stages were based on classification by Arosh et al. (2002), which correlates well with the goat specie classification as reported by Camp et al. (1983). Briefly, early stage CL are smaller in diameter (< 5 mm), profuse with blood, red in color, are soft, and the bordering tissue is not well defined. The mid stage CL are larger in diameter (6-7 mm) than the early stage, have a more flesh-like pink color, are more compact in consistency, and are entirely covered with a distinct connective tissue layer. The late stage CL are a little larger in diameter (7-10 mm) than the mid stage, are less pink in color, and have a distinct connective tissue layer cover. All does were housed outdoors in covered facilities (9.5 × 15.5 meter pens) and were observed twice daily for classical behavioral estrus using an intact male. To ensure normal cycle length, tissue collection did not take place until does were well within the breeding season, i.e. cycling began in late August and tissue collection occurred between the months of November and February. At estrus, and daily thereafter, blood samples were collected, via jugular venipuncture, until day of CL tissue collection (D3, D10, or D15) for analysis of serum progesterone for confirmation of normal luteal function. 2.2. Corpus luteum tissue harvest Does were fasted for 18-22 h and water was removed ~10 h prior to tissue collection. After the fasting period, does were transported to the surgical facility at the TAMUK farm where they were anesthetized using inhalation of isoflurane gas from a vaporizer set at 4%, mixed with an O2 at a flow rate of 4 L/min. After initial induction, the vaporizer was set to 2.5%-3% with an O2 flow rate of 2.8–3.2 L/min. A 3 to 4 inch vertical incision was made between the udder quarters to expose the reproductive tract. Ovaries were removed via blunt dissection and placed in ice-cold Hanks solution for transportation. Corpora lutea were
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harvested, divided, and processed for paraffin embedding, RNA extraction, and/or cell culture.
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2.3. Immunohistochemistry
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Luteal tissue (n=6 animals/luteal stage) was placed in ice-cold (4°C) fixative solution (10% Formalin, ~4% formaldehyde; Sigma, St. Louis, MO) for 24 h at 4°C on a shaker and then rinsed twice in 1x phosphate buffered saline (PBS) for 1 h each. Tissue was then placed in a graded series of ethanol baths (40% (v/v), 60%, 80%, 95% (2x), and 100% (2x) ethanol) for 1 h each at 4°C and two separate 1 h rinses in xylene. Dehydrated luteal tissue was transferred to a paraffin (TissuePrep; Fisher Scientific, Pittsburgh, PA) bath for 2 h and then embedded in a paraffin mold. Tissue was sectioned (6 μm) and placed on glass slides (2 serial sections per slide). Tissue was deparaffinized and rehydrated in xylene (2x) for 5 min each and rehydrated through a graded series of ethanol baths (100% (2x), 95% (2x), and 70%) for 2 min each, rinsed in tap water for 5 min, and incubated in 3% (v/v) hydrogen peroxide for 10 min to block endogenous peroxidase activity. Following deparaffinization and rehydration, sections were boiled in 10 mM sodium citrate for antigen recovery, cooled to room temperature, and rinsed in buffer (1% (w/v) bovine serum albumin (BSA) in PBS) for 5 min. Slides were processed as per manufacturer’s recommendations using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). To block nonspecific binding, tissue was incubated for 30 min at room temperature with blocking solution [1% (w/v) BSA and 0.3% (v/v) Triton-X-100 in PBS], and normal goat or horse serum (1% v/v; Vector Laboratories). Following a rinse in buffer, slides were incubated in 3% BSA in PBS for blocking at 37°C for 1 h. Following another buffer rinse, slides were incubated with Ang1 (1:50; CA0636; Cell Applications, San Diego, CA), FGF2 (1:50; CA0259; Cell Applications), VEGF (1:50; SC-7269; Santa Cruz Biotechnology Inc., Santa Cruz, CA), leptin (1:500; PA1-052; Thermo Scientific, Waltham, MA), or ObRb (1:50; SC-8325; Santa Cruz) primary antibodies in buffer. For each slide incubated with primary polyclonal antibody, a consecutive slide was incubated in normal serum (1.5% v/v) as a control. Slides were rinsed and incubated with biotinylated secondary antibody (0.5% v/v; Vector laboratories) for 30 min at room temperature. Following secondary antibody incubation, tissue sections were rinsed, incubated in an avidin biotinylated horseradish peroxidase complex (1% v/v) for 30 min at room temperature, rinsed, and incubated with peroxidase substrate solution (NovaRED Substrate Kit; Vector) for 10 min at room temperature. Each slide was rinsed, air dried, mounted in Permount medium and analyzed. To validate primary antibody binding anti-FGF2, anti-Ang1, anti-VEGF, and anti-leptin were pre-incubated with 1 μg of FGF2 (200-12; Shenandoah), Ang1 (100-46; Shenandoah Biotechnology, Inc., Warwick, PA), VEGF (10-663-45110; GenWay Biotech, San Diego, CA), leptin (CRL303B; Cell Sciences, Canton, MA), respectively, for 1 h and used during the primary antibody step. To verify anti-ObRb binding, sample slides were pre-incubated with leptin (Cell Sciences) for 1 h and rinsed then treated with antibody. Slides were observed at 20x for location of NovaRED staining, including small (5-20 μm) and large (>20 μm) luteal cells, and vessels (Kalender and Arikan, 2007).
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2.4. Dispersed corpora lutea cell cultures
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Luteal tissue (n=6 animals/luteal stage) was mechanically minced and enzymatically dissociated in digestion media (TypeIA collagenase, 1.27 mg/ml, 0.5% BSA in Dulbecco’s Modified Eagle’s Medium; DMEM) for 90 min at 37°C with gentle agitation in a shaking water bath. Cells were filtered through a metal mesh (80 μm) and centrifuged twice at 150 × g for 4 min and the enzymatic supernatant was removed. Cells were re-suspended and the numbers of viable cells were determined using the Trypan Blue Exclusion Test. Cell viability was greater than 85% for each luteal stage. The dispersed luteal cells were seeded in treated polystyrene 24-well cluster dishes (Sigma) at 3 × 106 cells/well in attachment serum media [1 ml/well; DMEM supplemented with 10% (v/v) charcoal stripped fetal calf serum and 5,000 μg/mL penicillin/streptomycin] and incubated for 24 h in a humidified atmosphere of 5% CO2 in air at 37°C to equilibrate cells to culture condition, allow aggregation, and promote adherence. Following the 24 h incubation, attachment serum media was aspirated and replaced with culture media (1 ml/well; DMEM, 0.1% (w/v) BSA, 2.0% (v/v) charcoal stripped fetal calf serum, 0.5 mM ascorbic acid, and 5,000 μg/mL penicillin/streptomycin) containing recombinant ovine leptin (0, 10−12, 10−11, 10−10, 10−9, 10−8 M; Cell Sciences) in triplicate (3 wells/dose/animal/luteal stage) for 24 h in a humidified atmosphere of 5% CO2 in air at 37°C. Ovine leptin, which is >95% identical, at the amino acid level, to caprine leptin (Genebank access. #Q28603 and #CAJ38273), was substituted due to availability. After 24 h, the cell culture media was aspirated and stored at −80°C for measurement of secreted progesterone as a marker for effect of leptin in vitro. Denaturing solution [1 ml/well; 25 mM sodium citrate (pH 7.0), 0.5% (w/v) Nlaurylsarcosine, 4 M guanidine thiocyanate with 0.7% (v/v) 2-mercaptoethanol] was added to cells, aspirated, pooled by dose, and stored at −80°C for total RNA extraction and mRNA analysis. 2.5. Gene expression
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Total RNA was extracted from CL tissue (10-20 mg) (early stage, n=12; mid stage, n=8; late stage, n=11) using a phenol:chloroform based procedure, as previously reported (Garcia et al., 2002), and analyzed for FGF2, Ang1, VEGF, leptin, and ObRb (tissue only) mRNA using real-time polymerase chain reaction (PCR) with the DNA Engine Opticon II (Bio-Rad, Hercules, CA). All enzymes and PCR reagents were obtained from Promega (Madison, WI). Two μg of total RNA was treated for DNA contamination in a reaction mix containing moloney-murine leukemia virus reverse transcriptase (M-MLV RT), reaction buffer (50 mM Tris-HCL (pH 8.3), 75 mM KCL, 3 mM MgCl2, and 10mM DTT), nucleotide mix (dATP, dCTP, dGTP, dTTP, 4 μM/μl), RNASIN (20-40 u/μl), RQ1 DNase (1 u/μl) and RNase free water in a total volume of 25 μl. Each reaction mix was incubated at 37°C for 30 min for DNase treatment, followed by an 85°C incubation for 10 min to terminate DNase activity. Following DNase treatment, Oligo dt15 (500 μg/ml) and M-MLV RT (200 u/μl) was added and incubated at 37°C for 1 h to RT mRNA into cDNA. Samples were then incubated at 85°C for 10 min to terminate the RT. Complementary DNA was amplified by real-time PCR using primers synthesized by Integrated DNA Technologies (Coraville, IA; Table 1) in a Takara SYBR® Green (Madison, WI) reaction mix. The VEGF primer pair was developed using the Bos taurus sequence for VEGFA mRNA (Genebank accession # NM_174216.1)
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and does not differentiate among the 121, 165, and 189 isoforms reported in the bovine (Berisha et al., 2000), ovine (Redmer et al., 1996), rodent and human (Ferrara, 2004) CL. After a 10 min hot start, the PCR cycles were as follows: 94°C for 30 s, anneal temperature of 52°C for 30 s, 68°C or 72°C (relative to primer pair; Table 1) for 60 s for 36-44 cycles, ending with a 72°C 10 min extension. Cyclophilin mRNA from porcine liver was used to create a standard curve for relative quantification purposes, a scientifically accepted method for real time quantification (Bustin, 2000) that has been previously published by our laboratory (Matsumoto et al., 2006). To correct for procedural variability, L19 (housekeeping gene) mRNA was used to normalize targeted amplicons. Relative values of FGF2, Ang1, VEGF, leptin, and ObRb gene expression were quantitated from the relative standard curve. Values were then transformed to log10 and normalized with L19. Cultured luteal cell values are represented as a percentage of the control (0M). Agarose gel electrophoresis, containing ethidium bromide, was used to confirm the presence of a single band of the expected size of the PCR amplicon for each of the PCR primer pair reactions. The Ang1, VEGF, and ObRb PCR amplicons were electrophoresed on a 2% agarose gel stained with ethidium bromide, extracted, purified and cloned using the pGEM®-T Easy Vector Systems (Promega). Cloned products were sequenced using MCLab Sequencing (San Francisco, CA) to verify the amplicon sequence as the correct target of the primers. 2.6. Radioimmunoassay Concentrations of progesterone in culture media and serum were determined by radioimmunoassay (RIA, Coat-A-Count®; Siemens Corp, New York, NY; Novak et al., 2003; Mao et al., 2001) to confirm cell viability during culture process, response to culture treatment (Fig. 4), and the presence of a functional CL at the time of tissue harvest. Progesterone concentrations were validated by the recovery of exogenous progesterone added in known amounts (1, 2.5, 5, 10, and 20 ng/mL) to pooled goat serum. Recovery of exogenous hormone was ≥100%. Inter- and intra-assay coefficients of variation for all assays were
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Published in final edited form as: Anim Reprod Sci. 2014 August ; 148(0): 121–129. doi:10.1016/j.anireprosci.2014.05.002.
The effect of leptin on luteal angiogenic factors during the luteal phase of the estrous cycle in goats Jessica R. Wiles1, Robin A. Katchko1, Elizabeth A. Benavides1, Chad W. O’Gorman1, Jean M. Escudero2, Duane H. Keisler3, Randy L. Stanko1,4, and Michelle R. Garcia1,* 1Department
of Animal, Rangeland & Wildlife Science, Dick and Mary Lewis Kleberg College of Agriculture, Natural Resources and Human Sciences, Texas A&M University-Kingsville, Kingsville, TX 78363 2Department
of Biological and Health Sciences, College of Arts and Sciences, Texas A&M University-Kingsville, Kingsville, TX 78363
NIH-PA Author Manuscript
3Division
of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, MO 65211
4Animal
Reproduction Laboratory, Texas A&M University AgriLife Research Station, Beeville, TX
78102
Abstract
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Fibroblast growth factor 2 (FGF2), angiopoietin 1 (Ang1), and vascular endothelial growth factor (VEGF) are angiogenic factors implicated in the vascular development of the corpus luteum (CL). Each factor is regulated or influenced by leptin in non-ovarian tissues. Moreover, leptin and its receptor, ObRb, have been identified in luteal tissue throughout the luteal phase. Therefore, leptin is hypothesized to influence luteal vasculature through the regulation of FGF2, Ang1, and VEGF. Multiparous, cycling crossbred female goats (does) were allocated to early (n=12), mid (n=8), and late (n=11) stages of the luteal phase for CL collection. Luteal tissue was harvested and either snap frozen in liquid N2, paraffin embedded, or cultured with leptin (0, 10−12, 10−11, 10−10, 10−9, 10−8 M). Tissue was analyzed for FGF2, Ang1, VEGF, ObRb, and leptin expression. Angiopoietin 1, FGF2, VEGF expression was higher (P≤0.001) in the mid-luteal stage than the early stage. Expression decreased (P≤0.001) during the late luteal stage with the exception of VEGF, which remained elevated. In contrast, leptin and ObRb were lowest (P≤0.003) during the mid-luteal stage compared to the early and late stages. All factors were detected in and/or around vessels in early stage tissue compared to mid and late stages. Leptin stimulated (P≤0.02) Ang1, FGF2, and VEGF
*
Corresponding Author: Texas A&M University-Kingsville, Department of Animal, Rangeland & Wildlife Sciences, Kleberg Ag. Bldg. Rm 133, Kingsville, TX 78363, USA. Tel: +1-361-593-3197; fax: +1-361-593-3788; [email protected]. Competing interest There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Authors’ contributions JRW participated in study design, acquisition, analysis and interpretation of data, and in preparation of the manuscript. RAK, EAB, and CWO participated in acquisition and interpretation of the data. JME, DHK, and RLS participated in the analysis of data. As Principle Investigator, MRG participated in the intellectual and experimental design of the study, data acquisition, analysis and interpretation, as well as writing and revising the manuscript. All authors read and approved the final manuscript. Publisher's Disclaimer: 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 citable 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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expression only in early stage luteal cultures. Collectively, these data provide evidence that leptin may be involved in the luteal angiogenic process during the early stage of CL formation.
NIH-PA Author Manuscript
Keywords leptin corpus luteum angiogenesis goats
1. Introduction
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Abnormal vasculature of the CL is a defect that leads to abnormal development and decreased progesterone production (Glock and Brumsted, 1995; Hazzard and Stouffer, 2000). Identifying and understanding the underlying mechanisms of luteal angiogenesis may lead to a reduction in luteal-related infertility and alternate methodology for the regulation of reproductive cycles. In the ovary, angiogenesis is a cyclical, recurring process that takes place following ovulation. Upon ovulation, the basement membrane between the granulosa and thecal layers disassociates allowing the thecal capillaries to invade the avascular granulosa layer and antral space forming a nascent network of capillaries to nourish the developing CL. The luteal neo-vascularization process is attributed to the biological activity of FGF2, Ang1, and VEGF. Both FGF2 and VEGF promote capillary membrane destabilization, endothelial cell differentiation, proliferation, migration, and vascular tube formation in human, bovine, and ovine luteal tissue (Suzuki et al., 1998; Reynolds and Redmer, 1998). Maturation and stabilization of nascent vessels is then promoted by Ang1 through the recruitment of stromal support cells, including pericytes and smooth muscle cells (Shalaby et al., 1995). Interestingly, each of these angiogenic regulatory factors is influenced by the adipogenic hormone leptin (Aleffi et al., 2005, Cao et al., 2001).
NIH-PA Author Manuscript
Both leptin and its receptor (ObRb) have been identified in normal and polycystic ovaries of various species (Loffler et al., 2001; Ruiz-Cortez et al., 2000; Ryan et al., 2003; MunozGutierrez et al., 2005), particularly in steroidogenically active follicles and luteal tissue. This would infer that leptin may be involved in steroidogenic processes (Kendall et al., 2004). However, in order to substantially modify steroid production cells must be incubated with leptin in the presence of a growth promotant (Spicer and Francisco 1997; Spicer and Francisco 1998; Zachow and Magoffin 1997; Karlsson et al., 1997; Brannian et al., 1999), which suggests that an ancillary role for leptin in ovarian tissue, such as angiogenesis, may exist. In support, ObRb has been identified in ovarian vascular endothelial cells in rodents (Ryan et al., 2003) and its expression in the porcine CL is highest during luteal development, a period of intense angiogenic activity (Ruiz-Cortez et al., 2000). Furthermore, leptin alone is capable of stimulating angiogenesis in the cornea of the eye (Sierra-Honigmann et al., 1998) and upregulates the expression of angiogenic factors in nonovarian tissue (Aleffi et al., 2005, Cao et al., 2001). Collectively, the evidence supports the supposition that leptin may be involved in ovarian angiogenesis, specifically vascularization of a developing CL. Therefore, it is hypothesized that leptin will influence the expression of angiogenic growth factors, FGF2, Ang1, and VEGF, in developing luteal tissue. The objectives were to: 1.) characterize FGF2, Ang1, VEGF, leptin, and ObRb expression at different stages of the luteal phase of the estrous cycle, and 2.) determine the effect of leptin
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on the expression of FGF2, Ang1, and VEGF in dispersed luteal tissue during the early, mid, and late stages of the luteal phase. Understanding the mechanisms of the development of luteal tissue may lead to a future reduction in the occurrence of infertility associated with luteal defects.
2. Materials and methods Institutional Animal Care and Use Committee at Texas A&M University-Kingsville (TAMUK) approved the animal care procedures used for this study. 2.1. Animals
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Thirty-one mature, cycling crossbred (Boer × Spanish) female goats (does), Capra aegagrus hircus, averaging 11 months of age and weighing 38.2 ± 2.3 kg at date of tissue collection, from the Texas A&M University-Kingsville (TAMUK) farm were utilized. Animals were randomly allocated for CL tissue collection at either the early (n=12), mid (n=8), or late (n=11) stages of the luteal phase of the estrous cycle. Early, mid, and late stages of the luteal phase are defined by the day of the estrous cycle beginning with day of initial onset of estrus (D0). Therefore, ‘early stage’ CL were collected on D3 of the estrous cycle which is ~35-42 h post-ovulation, while ‘mid stage’ CL were collected on D10 of the estrous cycle, and ‘late stage’ CL were collected on D15 of the normally 21-day caprine estrous cycle. Tissue was collected to satisfy minimum requirements of 6 does/luteal stage, not exceeding 12 does/ luteal stage, for tissue characterization analysis and cell cultures. The specifically defined luteal stages were based on classification by Arosh et al. (2002), which correlates well with the goat specie classification as reported by Camp et al. (1983). Briefly, early stage CL are smaller in diameter (< 5 mm), profuse with blood, red in color, are soft, and the bordering tissue is not well defined. The mid stage CL are larger in diameter (6-7 mm) than the early stage, have a more flesh-like pink color, are more compact in consistency, and are entirely covered with a distinct connective tissue layer. The late stage CL are a little larger in diameter (7-10 mm) than the mid stage, are less pink in color, and have a distinct connective tissue layer cover. All does were housed outdoors in covered facilities (9.5 × 15.5 meter pens) and were observed twice daily for classical behavioral estrus using an intact male. To ensure normal cycle length, tissue collection did not take place until does were well within the breeding season, i.e. cycling began in late August and tissue collection occurred between the months of November and February. At estrus, and daily thereafter, blood samples were collected, via jugular venipuncture, until day of CL tissue collection (D3, D10, or D15) for analysis of serum progesterone for confirmation of normal luteal function. 2.2. Corpus luteum tissue harvest Does were fasted for 18-22 h and water was removed ~10 h prior to tissue collection. After the fasting period, does were transported to the surgical facility at the TAMUK farm where they were anesthetized using inhalation of isoflurane gas from a vaporizer set at 4%, mixed with an O2 at a flow rate of 4 L/min. After initial induction, the vaporizer was set to 2.5%-3% with an O2 flow rate of 2.8–3.2 L/min. A 3 to 4 inch vertical incision was made between the udder quarters to expose the reproductive tract. Ovaries were removed via blunt dissection and placed in ice-cold Hanks solution for transportation. Corpora lutea were
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harvested, divided, and processed for paraffin embedding, RNA extraction, and/or cell culture.
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2.3. Immunohistochemistry
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Luteal tissue (n=6 animals/luteal stage) was placed in ice-cold (4°C) fixative solution (10% Formalin, ~4% formaldehyde; Sigma, St. Louis, MO) for 24 h at 4°C on a shaker and then rinsed twice in 1x phosphate buffered saline (PBS) for 1 h each. Tissue was then placed in a graded series of ethanol baths (40% (v/v), 60%, 80%, 95% (2x), and 100% (2x) ethanol) for 1 h each at 4°C and two separate 1 h rinses in xylene. Dehydrated luteal tissue was transferred to a paraffin (TissuePrep; Fisher Scientific, Pittsburgh, PA) bath for 2 h and then embedded in a paraffin mold. Tissue was sectioned (6 μm) and placed on glass slides (2 serial sections per slide). Tissue was deparaffinized and rehydrated in xylene (2x) for 5 min each and rehydrated through a graded series of ethanol baths (100% (2x), 95% (2x), and 70%) for 2 min each, rinsed in tap water for 5 min, and incubated in 3% (v/v) hydrogen peroxide for 10 min to block endogenous peroxidase activity. Following deparaffinization and rehydration, sections were boiled in 10 mM sodium citrate for antigen recovery, cooled to room temperature, and rinsed in buffer (1% (w/v) bovine serum albumin (BSA) in PBS) for 5 min. Slides were processed as per manufacturer’s recommendations using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). To block nonspecific binding, tissue was incubated for 30 min at room temperature with blocking solution [1% (w/v) BSA and 0.3% (v/v) Triton-X-100 in PBS], and normal goat or horse serum (1% v/v; Vector Laboratories). Following a rinse in buffer, slides were incubated in 3% BSA in PBS for blocking at 37°C for 1 h. Following another buffer rinse, slides were incubated with Ang1 (1:50; CA0636; Cell Applications, San Diego, CA), FGF2 (1:50; CA0259; Cell Applications), VEGF (1:50; SC-7269; Santa Cruz Biotechnology Inc., Santa Cruz, CA), leptin (1:500; PA1-052; Thermo Scientific, Waltham, MA), or ObRb (1:50; SC-8325; Santa Cruz) primary antibodies in buffer. For each slide incubated with primary polyclonal antibody, a consecutive slide was incubated in normal serum (1.5% v/v) as a control. Slides were rinsed and incubated with biotinylated secondary antibody (0.5% v/v; Vector laboratories) for 30 min at room temperature. Following secondary antibody incubation, tissue sections were rinsed, incubated in an avidin biotinylated horseradish peroxidase complex (1% v/v) for 30 min at room temperature, rinsed, and incubated with peroxidase substrate solution (NovaRED Substrate Kit; Vector) for 10 min at room temperature. Each slide was rinsed, air dried, mounted in Permount medium and analyzed. To validate primary antibody binding anti-FGF2, anti-Ang1, anti-VEGF, and anti-leptin were pre-incubated with 1 μg of FGF2 (200-12; Shenandoah), Ang1 (100-46; Shenandoah Biotechnology, Inc., Warwick, PA), VEGF (10-663-45110; GenWay Biotech, San Diego, CA), leptin (CRL303B; Cell Sciences, Canton, MA), respectively, for 1 h and used during the primary antibody step. To verify anti-ObRb binding, sample slides were pre-incubated with leptin (Cell Sciences) for 1 h and rinsed then treated with antibody. Slides were observed at 20x for location of NovaRED staining, including small (5-20 μm) and large (>20 μm) luteal cells, and vessels (Kalender and Arikan, 2007).
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2.4. Dispersed corpora lutea cell cultures
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Luteal tissue (n=6 animals/luteal stage) was mechanically minced and enzymatically dissociated in digestion media (TypeIA collagenase, 1.27 mg/ml, 0.5% BSA in Dulbecco’s Modified Eagle’s Medium; DMEM) for 90 min at 37°C with gentle agitation in a shaking water bath. Cells were filtered through a metal mesh (80 μm) and centrifuged twice at 150 × g for 4 min and the enzymatic supernatant was removed. Cells were re-suspended and the numbers of viable cells were determined using the Trypan Blue Exclusion Test. Cell viability was greater than 85% for each luteal stage. The dispersed luteal cells were seeded in treated polystyrene 24-well cluster dishes (Sigma) at 3 × 106 cells/well in attachment serum media [1 ml/well; DMEM supplemented with 10% (v/v) charcoal stripped fetal calf serum and 5,000 μg/mL penicillin/streptomycin] and incubated for 24 h in a humidified atmosphere of 5% CO2 in air at 37°C to equilibrate cells to culture condition, allow aggregation, and promote adherence. Following the 24 h incubation, attachment serum media was aspirated and replaced with culture media (1 ml/well; DMEM, 0.1% (w/v) BSA, 2.0% (v/v) charcoal stripped fetal calf serum, 0.5 mM ascorbic acid, and 5,000 μg/mL penicillin/streptomycin) containing recombinant ovine leptin (0, 10−12, 10−11, 10−10, 10−9, 10−8 M; Cell Sciences) in triplicate (3 wells/dose/animal/luteal stage) for 24 h in a humidified atmosphere of 5% CO2 in air at 37°C. Ovine leptin, which is >95% identical, at the amino acid level, to caprine leptin (Genebank access. #Q28603 and #CAJ38273), was substituted due to availability. After 24 h, the cell culture media was aspirated and stored at −80°C for measurement of secreted progesterone as a marker for effect of leptin in vitro. Denaturing solution [1 ml/well; 25 mM sodium citrate (pH 7.0), 0.5% (w/v) Nlaurylsarcosine, 4 M guanidine thiocyanate with 0.7% (v/v) 2-mercaptoethanol] was added to cells, aspirated, pooled by dose, and stored at −80°C for total RNA extraction and mRNA analysis. 2.5. Gene expression
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Total RNA was extracted from CL tissue (10-20 mg) (early stage, n=12; mid stage, n=8; late stage, n=11) using a phenol:chloroform based procedure, as previously reported (Garcia et al., 2002), and analyzed for FGF2, Ang1, VEGF, leptin, and ObRb (tissue only) mRNA using real-time polymerase chain reaction (PCR) with the DNA Engine Opticon II (Bio-Rad, Hercules, CA). All enzymes and PCR reagents were obtained from Promega (Madison, WI). Two μg of total RNA was treated for DNA contamination in a reaction mix containing moloney-murine leukemia virus reverse transcriptase (M-MLV RT), reaction buffer (50 mM Tris-HCL (pH 8.3), 75 mM KCL, 3 mM MgCl2, and 10mM DTT), nucleotide mix (dATP, dCTP, dGTP, dTTP, 4 μM/μl), RNASIN (20-40 u/μl), RQ1 DNase (1 u/μl) and RNase free water in a total volume of 25 μl. Each reaction mix was incubated at 37°C for 30 min for DNase treatment, followed by an 85°C incubation for 10 min to terminate DNase activity. Following DNase treatment, Oligo dt15 (500 μg/ml) and M-MLV RT (200 u/μl) was added and incubated at 37°C for 1 h to RT mRNA into cDNA. Samples were then incubated at 85°C for 10 min to terminate the RT. Complementary DNA was amplified by real-time PCR using primers synthesized by Integrated DNA Technologies (Coraville, IA; Table 1) in a Takara SYBR® Green (Madison, WI) reaction mix. The VEGF primer pair was developed using the Bos taurus sequence for VEGFA mRNA (Genebank accession # NM_174216.1)
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and does not differentiate among the 121, 165, and 189 isoforms reported in the bovine (Berisha et al., 2000), ovine (Redmer et al., 1996), rodent and human (Ferrara, 2004) CL. After a 10 min hot start, the PCR cycles were as follows: 94°C for 30 s, anneal temperature of 52°C for 30 s, 68°C or 72°C (relative to primer pair; Table 1) for 60 s for 36-44 cycles, ending with a 72°C 10 min extension. Cyclophilin mRNA from porcine liver was used to create a standard curve for relative quantification purposes, a scientifically accepted method for real time quantification (Bustin, 2000) that has been previously published by our laboratory (Matsumoto et al., 2006). To correct for procedural variability, L19 (housekeeping gene) mRNA was used to normalize targeted amplicons. Relative values of FGF2, Ang1, VEGF, leptin, and ObRb gene expression were quantitated from the relative standard curve. Values were then transformed to log10 and normalized with L19. Cultured luteal cell values are represented as a percentage of the control (0M). Agarose gel electrophoresis, containing ethidium bromide, was used to confirm the presence of a single band of the expected size of the PCR amplicon for each of the PCR primer pair reactions. The Ang1, VEGF, and ObRb PCR amplicons were electrophoresed on a 2% agarose gel stained with ethidium bromide, extracted, purified and cloned using the pGEM®-T Easy Vector Systems (Promega). Cloned products were sequenced using MCLab Sequencing (San Francisco, CA) to verify the amplicon sequence as the correct target of the primers. 2.6. Radioimmunoassay Concentrations of progesterone in culture media and serum were determined by radioimmunoassay (RIA, Coat-A-Count®; Siemens Corp, New York, NY; Novak et al., 2003; Mao et al., 2001) to confirm cell viability during culture process, response to culture treatment (Fig. 4), and the presence of a functional CL at the time of tissue harvest. Progesterone concentrations were validated by the recovery of exogenous progesterone added in known amounts (1, 2.5, 5, 10, and 20 ng/mL) to pooled goat serum. Recovery of exogenous hormone was ≥100%. Inter- and intra-assay coefficients of variation for all assays were
