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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
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* Department of Animal and Range Sciences,
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Center for Nutrition and Pregnancy and
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Cell Biology Center, North Dakota State University, Fargo 58105;
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Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand 40002; and and
|| Department of Animal Science, University of Wyoming, Laramie 82071
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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3 mm for determination of estradiol-17ß (E), progesterone (P4), vascular endothelial growth factor (VEGF), and IGFBP concentrations. The ovaries were then perfusion-fixed and used for histochemical detection of lectin BS-1 (a marker of endothelial cells and thus vascularization) binding, and immunolocalization of VEGF, endothelial nitric oxide synthase (eNOS), and proliferating cell nuclear antigen, followed by image analysis of selected follicles. Follicles were classified, based on E and P4 concentrations in FF, as dominant, estrogen-active (EA; E:P4 1) or nondominant, estrogen-inactive (EI; E:P4 <1). Concentrations of E and VEGF in FF, the area of positive staining for lectin BS-1, VEGF, and eNOS, and the labeling index (an index of the percentage of cells proliferating) in granulosa and theca layers were greater (P < 0.05) in the EA than in the EI follicles, but concentrations of P4 and IGFBP in FF were less (P < 0.05) in EA than in EI follicles. In addition, vascularity was positively correlated (P < 0.05) with VEGF and eNOS protein expression, and tended (P < 0.1) to be positively correlated with the E:P4 ratio in FF but tended (P < 0.1) to be negatively correlated with IGFBP and P4 concentrations in FF. These data highlight the importance of vascularity, angiogenic factors, and IGFBP in the health of the dominant follicle in heifers, and indicate that the FF concentrations of E, VEGF, IGFBP, and P4, and the E:P4 ratio can be used as markers of dominant follicles.
Key Words: cow • cell proliferation • dominant follicle • nitric oxide • vascularity • vascular endothelial growth factor
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Fortune et al., 2001). At each follicular wave, a cohort of follicles is initiated to grow, but only the dominant follicle continues to grow and has a great capacity to produce estradiol-17ß (E; Evans and Fortune, 1997; Mihm et al., 2002; Beg and Ginther, 2006).
Maintenance of follicular health depends on the presence of angiogenic factors and a functional vasculature (Hirshfield, 1991; Zeleznik, 2001; Jiang et al., 2003). It has been demonstrated that angiogenesis and the development of vascularity may influence maturation of the preovulatory follicle and selection of a dominant ovulatory follicle (Redmer and Reynolds, 1996; Augustin, 2001).
Several angiogenic factors, including vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS), are expressed in ovarian follicles of several species (Grazul-Bilska et al., 2001, 2006; Reynolds et al., 2002). It has been suggested that these 2 angiogenic factors, along with other intrafollicular factors, including E and the family of IGF and IGFBP, are involved in the regulation of follicular growth and selection of a dominant follicle (Acosta and Miyamoto, 2004; Fortune et al., 2004; Beg and Ginther, 2006). We hypothesized that vascularity, expression of angiogenic factors, and concentrations of selected factors in follicular fluid (FF) would differ in dominant follicles compared with nondominant follicles.
Therefore, the objectives of this study were to 1) determine the concentrations of E, progesterone (P4), VEGF, and IGFBP in FF; 2) characterize vascularization, expression of VEGF and eNOS proteins, and the labeling index (LI; an index of the percentage of cells proliferating); and 3) evaluate the relationships among these variables in estrogen-active (EA; potentially dominant) and estrogen-inactive (EI; potentially nondominant) follicles of the first follicular wave in cows.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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analogue (Estrumate, Animal Health Corp., Union, NJ), given 11 d apart. Estrus was checked twice daily by using an androgenized cow. The first day of estrus was designated as d 0 of the subsequent first follicular wave. The ovaries were collected at slaughter on d 3 after the onset of estrus, which corresponded to a time before the first day of dominance during the first follicular wave (Austin et al., 2001).
For each ovary, the surface diameter of all visible follicles was determined in 2 axes by using a ruler, and the diameter of all surface follicles 3 mm was recorded. The location of all follicles within an ovary was mapped, and FF was gently aspirated from all follicles 3 mm. Follicular fluid was centrifuged (1,800 x g) immediately and stored at –70°C until the concentrations of E, P4, VEGF, and IGFBP were determined. The ovaries were perfused through the main ovarian artery with 20 mL of PBS (0.01 M phosphate and 0.14 M NaCl, pH 7.3) and then with 3 to 5 mL of Evans blue dye (0.5 g/L) to determine the extent of perfusion, followed by a 10-min perfusion with Carnoy’s solution before immersion in Carnoy’s solution for 2 h. The ovaries then were cut transversely into 2 to 4 pieces (cross-sections), which were immersed in Carnoy’s solution for another 2 h at room temperature. Fixed ovarian pieces were dehydrated by using a graded series of ethanol, cleared with a histological clearing agent (Histo-Clear, National Diagnostics, Atlanta, GA), paraffin-embedded, sectioned at 4 µm, and mounted onto glass slides, as previously described (Redmer et al., 2001).
E and P4 RIA
Concentrations of E and P4 were determined in unextracted FF diluted (1:20 to 1:225) with gel-PBS (0.01 M phosphate, 0.14 M NaCl, and 0.1% gelatin, pH 7.4; Redmer et al., 1991). Sensitivity of these assays was 1 pg/mL for E and 0.25 ng/mL for P4. For each hormone, FF samples were analyzed in a single assay, and the intraassay CV was 2.7% for E and 3.8% for P4.
Classification of Follicles
The ratio of E:P4 concentrations in FF was used to classify individual follicles as EA (E:P4 1; potentially dominant) or EI (E:P4 <1; potentially nondominant) follicles (Redmer et al., 1991; Sunderland et al., 1994). For each of the 8 heifers, selected follicles from the cohort were chosen for further evaluation, as described in Table 1. In heifer 1, of the 17 EI follicles, the largest follicle (15 mm in diameter) was the only one used for further evaluations. In heifer 2, a single follicle was clearly larger than the remaining follicles in the cohort (10 mm vs. 4 to 6 mm in diameter) and was EA; this follicle was therefore the only one used for further evaluations. In the remaining 6 heifers, 2 to 4 follicles were chosen for further evaluations based on the first criteria that they were the largest or second-largest follicles; of these follicles, 13 were EA and 3 were EI. Thus, a total of 14 EA and 4 EI follicles were evaluated (Table 1) across all heifers. The remaining follicles (n = 6 to 16 per heifer) averaged 6 mm in diameter and had an E:P4 ratio of 1.35, and were not used for further evaluations.
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). Briefly, human recombinant VEGF165 (cold hormone; G143AB, Genentech Inc., Los Angeles, CA), primary antibody (polyclonal rabbit antiserum to VEGF165; cat. no. 27906-17, Genentech Inc.), and human recombinant [125I]VEGF165 (tracer; NEX328, NEN Life Science Products Inc., Boston, MA) were used. All samples were analyzed in triplicate in a single assay, and the intraassay CV was 15.5%. The sensitivity averaged 25 pg/mL, and was defined as the VEGF standard yielding 95% of the counts in the buffer control tube. Intraassay variability for VEGF was determined by assaying a pool of ovine FF to which known quantities of VEGF had been added (0.0, 0.5, and 5.0 ng/mL of FF). The resulting concentrations (±SEM), after subtraction of the FF blank, averaged 0.81 ± 0.10 and 4.59 ± 0.20 ng/mL, respectively.
Ligand Blot Analysis of IGFBP
This assay was performed to determine the relative concentration of total IGFBP in FF, measured in optical density units (ODU). Human recombinant IGF-I (5 µg; GF-050-4, Austral Biologicals, San Ramon, CA) was iodinated with Chloramine-T. Iodinated IGF-I was purified on an anion-exchange resin (50 to 100 dry mesh, chloride form; AG 1-X8, Bio-Rad Laboratories, Hercules, CA) that was previously equilibrated with column buffer (0.05 M Na2HPO4, 3% BSA, pH 7.2). Specific activity was 17.8 µCi/µg of protein. Radiolabeled IGF-I was diluted to 200,000 cpm [0.1 µCi/mL in Tris-buffered saline (TBS)-Tween (10 mM Tris, 0.15 M NaCl, 0.05% NaN3, 0.1% Tween-20, in 1% BSA)] before ligand blot analysis was performed (Mihm et al., 2000). All samples of FF (20 µg of protein/dot) were dot-blotted onto one Immobilon-P membrane (Millipore Corp., Bedford, MA) that had been prewetted with methanol and were then equilibrated with 5 washes of distilled water. As a positive control, 80 ng of human recombinant IGFBP4 (BP-360-2, Austral Biologicals) was dot-blotted onto the same membrane. The Immobilon-P membrane was dried and then rewetted with methanol and water before blocking for 4 h in 1% Blotto in TBS reagent (Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was incubated with radiolabeled IGF-I at 4°C for 18 h, washed in TBS-Tween and then TBS, covered with transparent wrap, and exposed to X-ray film (Kodak X-5, Sigma Chemical Co., St. Louis, MO) for 160 h at –70°C. Densitometry was performed with a densitometer (Model PDSI, Molecular Dynamics, Sunnyvale, CA) to determine the ODU for each sample. A greater value of ODU indicated a greater concentration of IGFBP in FF.
Histochemistry-Immunohistochemistry in Ovarian Tissues
Histochemical Localization of Lectin BS-1 Binding.
Detection of lectin BS-1 (from Bandeiraea simplicifolia, a marker of endothelial cells; Sigma) binding was performed as previously described (Redmer et al., 2001; Vonnahme et al., 2006). Briefly, to localize endothelial cells, ovarian tissue sections were incubated with biotin-labeled lectin BS-1 (10 µg/mL) overnight at 4°C followed by a wash in PBS and a 1-h incubation with Avidin:Biotinylated Enzyme Complex (ABC, Vector Laboratories, Burlingame, CA). For color development, all sections were incubated with SG substrate (Vector Laboratories) for 5 min. Tissue sections were then counterstained with fast red to visualize the nuclei. For controls, sections were incubated without biotin-labeled lectin BS-1. Vascularity was reported as the percentage of the total area that exhibited positive staining for lectin BS-1.
Immunohistochemical Localization of VEGF, eNOS, and Proliferating Cell Nuclear Antigen
Detection of VEGF, eNOS, and proliferating cell nuclear antigen (PCNA) was performed as previously described by Redmer et al. (2001), Grazul-Bilska et al. (2006), and Zheng et al. (1994), respectively. Briefly, ovarian tissue sections were deparaffinized, rehydrated, and incubated with 3% H2O2 in methanol to eliminate endogenous peroxidase activity. The sections were then rinsed several times in PBS containing Triton X-100 (0.3%, vol/vol) and, to block nonspecific binding of antibodies, were treated for 20 min with PBS containing either normal goat serum (1 to 2%, vol/vol) for further VEGF staining or normal horse serum (3%, vol/vol; ABC kit, Vector Laboratories) for further eNOS and PCNA staining.
The sections were incubated overnight at 4°C in PBS containing a primary antibody as follows: for VEGF, affinity-purified anti-VEGF rabbit serum (Red-1; Redmer et al., 1996) raised against a VEGF peptide (1:50 dilution); for eNOS, monoclonal eNOS/NOS Type III antibody (1:500 dilution; Transduction Laboratories, Lexington, KY; Grazul-Bilska et al., 2006); and for PCNA, monoclonal mouse antibody (1:500 dilution; MAB24R, Chemicon International, Temecula, CA; Zheng et al., 1993). Primary antibody was detected by using a biotin-labeled secondary antibody (antirabbit antibody for VEGF and antimouse antibody for eNOS and PCNA; Vector Laboratories) and the ABC method. For color development, SG substrate was used as described above. For VEGF controls, the primary antibody was replaced with normal rabbit IgG (diluted 1:100), and for eNOS and PCNA controls, the primary antibody was replaced with normal mouse IgG (4 µg/mL). After immunostaining, the tissue sections were counter-stained with nuclear fast red to visualize the nuclei.
Image Analysis
Images were taken from granulosa and theca layer areas of mapped EA and EI follicles. To determine changes in vascularity, angiogenic factor protein expression, and LI, the percentage of the total area that exhibited positive staining for lectin BS-1 (occupied by endothelial cells), VEGF or eNOS, the number of PCNA-positive (proliferating) cells, and the total number of cells per area were determined quantitatively by using an image analysis system (Image-Pro Plus, Media Cybernetics, Silver Spring, MD) as described previously (Grazul-Bilska et al., 2006; Vonnahme et al., 2006). For each follicle, 6 to 10 randomly chosen fields (0.025 mm2 per field) were evaluated separately for granulosa or theca layers. The data were expressed as the mean percentage ± SEM of the total area that exhibited positive staining within each field. The LI was calculated as a percentage (%) of proliferating cells out of the total number of cells within each tissue area. The LI for theca cells included all labeled cells, and no attempt was made to distinguish between theca cell types within the theca layer.
Statistical Analyses
The data were analyzed by GLM procedures (SAS Inst. Inc., Cary, NC). When the F-test was significant (P < 0.05), differences among means were evaluated by the Bonferroni t-test (Kirk, 1982). Means were considered different when P < 0.05, unless otherwise stated. Simple linear correlations between specific variables were determined by using PROC CORR of SAS.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Lectin BS-1 binding (a marker of endothelial cells and thus vascularity), and VEGF and eNOS proteins were detected in the theca layer of EA and EI follicles (Figure 1, panels A to F) and in blood vessels of ovarian stromal tissues (not shown). In theca of EA follicles, staining for lectin BS-1 binding, VEGF, and eNOS was strong and restricted to blood vessels (Figure 1, panels A, C, and E), but in EI follicles was weak and in some sections was not detectable (Figure 1, panels B, D, and F). Lectin BS-1 binding, VEGF, and eNOS were not detected in granulosa cells of EA or EI follicles. Positive PCNA staining was detected in the granulosa and theca layers of EA and EI follicles (Figure 1, panels G and H). Thecal vascularity, VEGF and eNOS protein expression, and the LI of granulosa and theca cells were greater (P < 0.01 to 0.05) in the EA than in the EI follicles (Table 3).
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shows correlation coefficients between specific variables of follicles evaluated. For all follicles evaluated, vascularity was positively correlated with VEGF and eNOS protein expression and with the E:P4 ratio in FF, but was negatively correlated with P4 and IGFBP concentrations in FF. Expression of VEGF protein was positively correlated with VEGF concentration in FF, eNOS protein expression, and the LI of granulosa cells, but was negatively correlated with P4 and IGFBP concentrations in FF. Expression of eNOS protein was positively correlated with VEGF protein expression and granulosa cell LI, but was negatively correlated with P4 and IGFBP concentrations in FF. Progesterone concentration in FF was positively correlated with IGFBP concentration in FF and follicle size, but was negatively correlated with VEGF concentration in FF and with VEGF and eNOS protein expression and LI of granulosa and theca cells. Concentration of IGFBP in FF was positively correlated with P4 concentration in FF and with follicle size, but was negatively correlated with E:P4 ratio and VEGF concentration in FF, VEGF and eNOS protein expression, and the LI of granulosa and theca cells. Concentration of VEGF in FF was positively correlated with VEGF protein expression and the LI of granulosa and theca cells, but was negatively correlated with P4 and IGFBP concentrations in FF. The ratio of E:P4 in FF was positively correlated with E concentration in FF and the LI of granulosa and theca cells, but was negatively correlated with IGFBP in FF.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Reynolds et al., 2002). Thus, expression of angiogenic factors in the EA follicles may enhance recruitment of a vascular supply and contribute to maintaining these follicles in a nonatretic state, and may subsequently contribute to their selection to become the dominant follicle. In fact, EI follicles exhibited reduced expression of VEGF and eNOS as well as reduced vascularity and LI of granulosa and theca cells, despite the fact that they were larger than the EA follicles. Thus, across all follicles, vascularity was positively correlated with VEGF and eNOS protein expression and the E:P4 ratio in FF, but not with follicular diameter.
Increased vascularity in the EA follicles, as observed in our study, results in a greater vascular surface area for exchange of transcapillary nutrients, gonadotropins, growth factors, and other factors. In a monkey model, Zeleznik et al. (1981) demonstrated that the density of capillaries surrounding a healthy maturing follicle is greater than that of other smaller follicles, and that this density of capillaries is associated with increased delivery of gonadotropins to the maturing follicle, suggesting that angiogenesis may play a role in the development of the preovulatory follicle. In fact, establishment of a vascular network around developing follicles may be the rate-limiting step in the process of selecting the dominant follicle. In turn, an insufficient vascular supply could act as the trigger that leads to follicular atresia (Watson and Alzi’abi, 2002; Feranil et al., 2005). Thus, selection of the dominant follicle may depend on the formation of a rich vascular network and vascular permeability of the follicle, likely controlled by enhanced expression of VEGF and eNOS proteins, as observed in the current study. Alternatively, production of angiogenic factors by follicular cells may stimulate vascular development in the dominant follicle (Redmer et al., 1991).
In the current study, the size of the EA follicles was less than the size of the EI follicles, which differs from other reports showing that the future dominant follicle was slightly larger than others in the cohort (Ginther et al., 1997; Mihm et al., 2000). However, Fortune et al. (2001) indicated that the difference in size was not always predictive of future dominance. Thus, size of the follicle at d 3 of the estrous cycle may not always be the distinguishing factor between dominant and subordinate follicles during the first follicular wave in cows.
The current data demonstrated a greater E concentration in FF of EA follicles, compared with EI follicles, which indicates that EA follicles have a greater ability to produce E during a period of relatively low serum gonadotropin concentration. Estradiol-17ß is recognized as the follicular growth, differentiation, and survival factor, which enhances aromatase activity, promotes expression of LH receptors, and increases sensitivity of granulosa cells to FSH and LH (Rosenfeld et al., 2001; Beg et al., 2002; Quirk et al., 2004). The cessation of E production is one of the earliest events in the atretic process (Xu et al., 1995). A recent study revealed that the decrease in E production observed during atresia of the dominant follicle was the direct result of decreased activity of the aromatase enzyme within granulosa cells (Valdez et al., 2005). Increased E secretion, along with production of mitogenic activity by granulosa cells and the presence of angiogenic factors in FF of the EA follicles, likely enhance recruitment and maintenance of a vascular supply, thereby contributing to maintaining future dominant follicles in a nonatretic state (Redmer et al., 1991).
The greater E concentration in FF in EA than in EI follicles is likely associated with changes in the IGF system, which consists of IGF-I and IGF-II and of several IGFBP and IGFBP proteases (Spicer, 2004). In fact, the IGF system in dominant follicles is well recognized as a critical determinant of follicle fate, and availability of free IGF is believed to be a key factor for a follicle to become dominant (Fortune et al., 2004). Furthermore, IGF-I and IGF-II have been demonstrated to play an important role in stimulating granulosa cell proliferation and synergizing with gonadotropins to promote E production and differentiation of follicular cells (Spicer and Echternkamp 1995; Poretsky et al., 1999). However, IGF effects can be blocked by IGFBP (Beg and Ginther, 2006). Therefore, changes in IGFBP and free IGF concentrations in FF are associated with follicular growth, atresia, or stage of deviation (Mihm et al., 2000; Spicer, 2004). In our study, greater concentrations of IGFBP in FF were detected in EI than in EA follicles, which is consistent with results reported by others (Rivera and Fortune, 2003; Beg and Ginther, 2006). Therefore, our data support the concept that increased concentrations of IGFBP in FF indicate that a follicle is nondominant; thus, follicular IGFBP level can be used as a marker of follicular dominance (Stewart et al., 1996; Mihm et al., 2000).
In the present study and in previous studies (Redmer et al., 2001; Grazul-Bilska et al., 2006), VEGF and eNOS were localized to the theca blood vessels in ruminant ovaries, and intense staining of VEGF and eNOS was present in bovine EA follicles, but was much weaker or nonexistent in EI follicles. However, these angiogenic factors have been detected by others within both granulosa and theca cells of bovine follicles as well (Berisha et al., 2000; Greenaway et al., 2004; Isobe et al., 2005). Both VEGF and eNOS have been implicated as regulators of follicular development (Hunter et al., 2004; Fraser, 2006; Grazul-Bilska et al., 2006). In fact, VEGF has been demonstrated to regulate follicular growth directly, because inhibition of VEGF during the follicular phase interrupted preovulatory follicular development in several species (Zimmerman et al., 2001; Abramovich et al., 2006; Fraser, 2006). In other studies, VEGF concentration in FF increased significantly, reaching high levels in bovine preovulatory follicles, suggesting that VEGF is a major angiogenic factor that is involved in regulation of the proliferation of capillaries accompanying the selection of the preovulatory follicle, supporting the growth of the dominant follicle (Berisha et al., 2000). Furthermore, enhanced expression of VEGF in the EA follicle was associated with enhanced E concentration in FF in the current study. Others also demonstrated a positive correlation between VEGF and E concentrations in the FF in pigs (Mattioli et al., 2001). Thus, our study supports the concept that follicular VEGF plays a role in vascularization during follicular deviation (Beg and Ginther, 2006). We postulate, based on the above observations, that VEGF interacts with several extra- and intrafollicular factors during the process of follicular selection to regulate vascular development and permeability, and thus delivery of regulatory factors and nutrients to potentially dominant follicles.
In this study, we demonstrated that eNOS protein expression was correlated with VEGF protein expression. Additionally, a greater nitric oxide concentration in FF of preovulatory follicles than nonpreovulatory follicles has been reported in mares (Pinto et al., 2003). Endothelial nitric oxide synthase is recognized as having both angiogenic and strong vasodilatory effects, which can affect follicular development, steroidogenesis, and oocyte maturation (Jablonka-Shariff and Olson, 2000; Reynolds et al., 2002; Grazul-Bilska et al., 2006). Although our data and the data of others indicate that the nitric oxide system is involved in the regulation of follicular function, the specific role of the nitric oxide system in follicular selection has not been elucidated and requires further study.
Cellular proliferation in the granulosa and theca layers was greater in EA than in EI follicles in our study. A similar pattern of cell proliferation was reported for transitional and preovulatory follicles in mares (Watson and Alzi’abi, 2002). Furthermore, a greater proliferation rate in healthy than in atretic follicles was observed in sheep and cows (Jablonka-Shariff et al., 1994, 1996; Isobe and Yoshimura, 2000). This indicates that a significant decrease in granulosa and theca cell proliferation occurs in the EI follicles before or during selection of the dominant follicle. Such high proliferation of granulosa and theca cells of EA follicles may be closely associated with increased E concentrations in FF observed in our study, as mentioned above. Further, because it has been demonstrated that E can stimulate IGF-I production by granulosa cells, a factor that also affects follicular cell proliferation, it is very likely that these 2 factors, and possibly others such as VEGF, enhance granulosa and theca cell proliferation in EA, potentially dominant follicles.
In summary, this study demonstrated 1) greater concentrations of E and VEGF and a greater E:P4 ratio but lower concentrations of P4 and IGFBP in the FF of EA than of EI follicles, 2) immunolocalization of VEGF and eNOS in blood vessels of the theca of EA and EI follicles, 3) greater vascularization and expression of VEGF and eNOS proteins along with a greater LI in granulosa and theca cells of EA than of EI follicles, and 4) positive correlations between vascularity and the E:P4 ratio in FF, and between VEGF and eNOS expression in the theca, and negative correlations between vascularity and P4 and IGFBP concentrations in FF of follicles from the first follicular wave in cattle. This indicates that development of the vascular bed and locally enhanced expression of angiogenic factors play a role in follicle selection in cows. In addition, concentrations of E, P4, VEGF, and IGFBP, and the E:P4 ratio in FF can be used as markers of follicular health and atresia. Thus, these data provide new insight into the process of selection of a dominant follicle.
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Footnotes |
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2 Corresponding author: Dale.Redmer{at}ndsu.edu
Received for publication March 12, 2007. Accepted for publication April 26, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-induced regression in sheep. Reproduction 131:1115–1126.-hydoxylase, and cytochrome P450 aromatase in bovine follicles during the first follicular wave. Endocrinology 136:981–989.[Abstract]This article has been cited by other articles:
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  A. T Grazul-Bilska, J. S Caton, W. Arndt, K. Burchill, C. Thorson, E. Borowczyk, J. J Bilski, D. A Redmer, L. P Reynolds, and K. A Vonnahme Cellular proliferation and vascularization in ovine fetal ovaries: effects of undernutrition and selenium in maternal diet Reproduction, April 1, 2009; 137(4): 699 - 707. [Abstract] [Full Text] [PDF]
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