Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Binelli, M.
Right arrow Articles by Thatcher, W. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Binelli, M.
Right arrow Articles by Thatcher, W. W.
Agricola
Right arrow Articles by Binelli, M.
Right arrow Articles by Thatcher, W. W.
Biology of Reproduction 61, 127-134 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Persistent Dominant Follicle Alters Pattern of Oviductal Secretory Proteins from Cows at Estrus1

Mario Binellia, James Hampton3,a, William C. Buhib, and William W. Thatcher2,a

a Departments of Dairy and Poultry Sciences and b Obstetrics and Gynecology, University of Florida, Gainesville, Florida 32611-0920


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The experimental objective was to compare synthesis of oviductal secretory proteins of dairy cows bearing a persistent dominant follicle (PDF) versus a fresh dominant follicle (FDF) at estrus. On Day 7 after synchronized estrus (Day 0), cows received an intravaginal progesterone device and injection of prostaglandin F2{alpha} (PGF2{alpha}). On Day 9, cows received an injection of a GnRH agonist (FDF group; n = 3) or received no injection (PDF group, n = 3). On Day 16, all cows received PGF2{alpha}, and progesterone devices were removed. At slaughter on Day 18 or Day 19, oviducts ipsilateral and contralateral to the dominant follicle were divided into infundibulum, ampulla, and isthmus regions. Explants from oviductal regions were cultured in minimal essential medium supplemented with [3H]leucine for 24 h. Two-dimensional fluorographs of proteins in conditioned media were analyzed by densitometry. Rate of incorporation of [3H]leucine into macromolecules was greater in the infundibulum, ampulla, and isthmus of FDF cows (p < 0.01). Overall, intensities of radiolabeled secretory protein (P) 2 and P13 were greater for FDF than for PDF. In the ampulla, P14 was more intense for FDF while P7 was more intense for PDF. Abundance of P1 in the isthmus was greater for PDF cows. Across regions, P5, P6, P8, P9, and P11 were more intense for PDF than for FDF in the ipsilateral side. In the contralateral side, P19 was more intense for PDF than for FDF, whereas P6, P8, P9, and P11 were more intense for FDF. Differences in biosynthetic activity and in secreted oviductal proteins from cows bearing a PDF may contribute to the decrease in fertility associated with a PDF.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Synchronization of the estrous cycle in cattle is an important tool for reproductive management. For example, synchronization systems are used widely for artificial insemination, timed insemination, and embryo transfer. Most commonly, synchronization is achieved with combinations of treatments with prostaglandin F2{alpha} (PGF2{alpha}), progestins, and GnRH [1]. Synchronization with progestins is based on the principle that exogenous progestins, such as progesterone delivered by a controlled internal drug release (CIDR) device, can maintain a subluteal concentration of progestin in blood during a period that permits corpus luteum (CL) regression. In the absence of a CL, removal of the progestin source will result in a synchronized estrus [2].

During the estrous cycle in cattle, two to three follicular waves of dominant follicle development occur [3, 4]. Each follicular wave comprises periods of recruitment, selection, dominance, and turnover or atresia. The ovulatory follicle generated in the last wave does not turn over but ovulates in a low-progesterone (P4) environment. Turnover of the dominant follicle (DF) is associated with high concentration of P4, typical of mid-cycle, which lowers LH pulse frequency [5]. Turnover of the first-wave DF can be blocked by exogenous progestins and injection of PGF2{alpha} [6–8]. The resulting subluteal concentration of progestin in plasma permits an increase in LH pulse frequency, which sustains growth of the DF. This "persistent" DF (PDF) is estrogenic, and subsequent fertility, as measured by conception rate at first service (number of pregnancies per number of animals inseminated), is lower than that in animals bearing normal DFs (37.1% vs. 64.8% in heifers [7]; 23.6% vs. 58.2% for cows and heifers [8]). Fertility after artificial insemination, however, is restored to levels comparable to those of controls if PDF is turned over and a freshly recruited follicle is allowed to ovulate. Possible explanations for reduced fertility include alterations in the oocyte and/or in the oviductal environment. In a study by Ahmad et al. [9], embryos recovered from cows bearing PDF at Day 6 of pregnancy were less developed (i.e., were less able to reach the 16-cell stage) than embryos from cows ovulating fresh (F) DF. In addition, Revah and Butler [10] showed that oocytes recovered from PDF had expanded cumulus cells and condensed chromatin dispersed in their ooplasm. In contrast, compact cumulus cells and intact germinal vesicles were found in oocytes from FDF. Thus, the PDF may affect oocyte maturation or oviduct function, which could affect early embryonic development and decrease fertility.

Macromolecules present in oviductal fluid have been suggested to serve an important role in sperm capacitation [11], fertilization [12], and early embryo development [13]. Therefore, alterations in oviductal biosynthetic activity and protein synthesis and secretion may affect conception rate.

Steroid modulation of oviductal synthesis and secretion of proteins has been characterized in sheep [14, 15], baboons [16], and swine [17, 18]. An altered steroid environment, associated with development of a PDF, may alter oviductal protein synthesis and secretion. In turn, the altered pattern of protein synthesis and secretion could affect optimal oviductal function, fertilization, and early embryo development, and thereby increase embryonic death (i.e., reduce conception rate) in synchronized cows. The present experiment tested the hypothesis that the presence of a PDF alters protein synthesis and secretion of oviductal explants at estrus.

Specific objectives were 1) to induce a PDF or an FDF with the strategic use of PGF2{alpha}, a P4-containing CIDR, and GnRH; and 2) to compare the biosynthetic activity and the array of secretory proteins synthesized in the infundibulum (INF), ampulla (AMP), and isthmus (IST) at estrus in oviducts ipsilateral (IPSI) and contralateral (CONTRA) to the DF of cows bearing a PDF versus a FDF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Impervo paint was from Benjamin Moore and Co. (Jacksonville, FL), and the All-weather Paintstick was from LA-CO Industries, Inc./Markal Company (Chicago, IL). Donations of Lutalyse were made by Pharmacia-Upjohn Co. (Kalamazoo, MI), Buserelin was from Hoechst-Roussel Agri-Vet (Somerville, NJ) and CIDR-B devices were donated by EAZI-BREED, InterAg (Hamilton, New Zealand). Minimum essential medium Eagle (MEM), nonessential amino acids, anti-mycotic/antibiotic solution, and MEM vitamin solution were from Life Technologies (Gibco Laboratories, Grand Island, NY). L-[4,5-3H]Leucine (159 Ci/nmol) was from Amersham Life Sciences, Inc. (Arlington Heights, IL), and L-leucine, L-methionine, L-glutamine, D(+) glucose, bovine pancreatic insulin, riboflavin, and molecular weight standards were purchased from Sigma Chemical Co. (St. Louis, MO). Spectra/por 3 dialysis membrane was from Spectrum Medical Industries Inc. (Houston, TX). Acrylamide, N,N'-methylenebisacrylamide, SDS, Nonidet-P40, urea, agarose, and diallyltartardiamide were from BDH Laboratory Supplies (Poole, Dorset, UK). Ampholines were from Pharmacia (Uppsala, Sweden); N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulphate were from Bio-Rad (Hercules, CA). Glycine was from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Coomassie brilliant blue, fast green, bromophenol blue, ß-mercaptoethanol, hydrochloric acid, sodium hydroxide, Tris (hydroxymethyl) aminomethane, sodium salicylate, acetic acid, and chromatography paper were from Fisher Scientific (Fairlawn, NJ); and X-OMAT x-ray film was from Eastman Kodak Co. (Rochester, NY).

Animals and Treatment

During the pretreatment period, estrous cycles of six mature nonlactating cows were synchronized (Fig. 1). A used CIDR device containing approximately 1.2 g [19] of P4 was placed into the vagina of each cow for 7 days. One day before CIDR removal, cows received an injection of PGF2{alpha} (Lutalyse, 25 mg) to regress the CL. To aid with estrus detection, tail heads were painted (Impervo) and chalked (All-weather Paintstick). Cows were observed twice daily for signs of estrus, and paint scores were assigned [20]. The day of standing estrus was designated experimental Day 0. During the treatment period, ovaries were examined by transrectal ultrasonography using an Aloka echo camera model SSD 500 linear array ultrasound scanner equipped with a 7.5 MHz transducer (Aloka Co., Tokyo, Japan). From Days 5 to 18, follicles and CL were measured daily, and sizes were recorded. In addition, blood samples were collected in heparinized evacuated tubes (Vacutainers; Becton, Dickinson Vacutainer System USA, Rutherford, NJ) by tail venipuncture and stored in an ice bath. Plasma was harvested by centrifugation (1800 x g for 30 min) and stored at -20°C until assayed for estradiol-17ß (E2)and P4. On Day 7, all cows received injections of PGF2{alpha} and received one used CIDR device [7]. On Day 9, cows were assigned randomly to one of two treatment groups: cows of the FDF group received an injection of GnRH agonist (Buserelin, 8 µg) to induce turnover of any large follicles present at that time and allow recruitment of fresh follicles [21]. Cows of the PDF group did not receive the GnRH agonist. On Day 16, CIDR devices were removed, and cows received an injection of PGF2{alpha} (25 mg). Cows were checked for signs of estrus twice daily and slaughtered when observed in standing estrus (Day 18 or 19). In previous reports, the experimental models for persistent and fresh follicles resulted in a greater pregnancy rate for heifers inseminated at estrus induced by FDF [7, 21].



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 1. Experimental protocol (see text).

Tissue Culture

On the day of slaughter, reproductive tracts were removed aseptically, and oviducts were identified as IPSI or CONTRA to DF, dissected, trimmed free of mesosalpinx, and divided into INF, AMP, and IST regions on the basis of gross anatomical characteristics. Segments of tissue between IST and AMP were discarded. Tissue from each region was cut longitudinally to expose the lumen, and then minced into fragments of ~50 mm3. Tissue fragments from each functional region were cultured [18] in leucine-deficient minimal essential medium supplemented with [3H]leucine in the ratio of 100 mg tissue:3 ml medium:20 µCi [3H]leucine for 24 h at 37°C in a controlled atmosphere of N2:O2:CO2 (50%:47.5%:2.5% by volume). For AMP and INF, 500 mg of tissue was cultured per dish, while for IST variable amounts of tissue (between 140 and 290 mg) were used.

Two-Dimensional Electrophoresis

After 24-h incubations, conditioned media were dialyzed extensively (molecular weight cut-off 3500) against Tris-buffered saline (10 mM Tris, 150 mM NaCl) pH 7.6 (two changes of 4 L each/24 h) and then dialyzed against deionized water (two changes of 4 L each/24 h). Radioactivity in the retentate was determined by liquid scintillation spectrometry, and incorporation rate was defined as disintegrations per minute nondialyzable macromolecules per milligram of wet tissue. For each sample, a volume of dialyzed conditioned medium containing 4 x 105 dpm was lyophilized and submitted for two-dimensional (2D) SDS-PAGE as described by Buhi et al. [14]. Gels were stained with Coomassie blue dye, soaked in 1 M Na salicylate solution, dried, and exposed to x-ray film for 35 days at -80°C.

Densitometry

Fluorographs were developed, and after qualitative analysis, 20 protein spots were selected and analyzed quantitatively by densitometry (AlphaImager 2000; Alpha Innotech Corporation, San Leandro, CA). Since a constant amount of disintegrations per minute was loaded for all samples, the capacity of tissues to synthesize and secrete macromolecules (dpm/mg of tissue) was not accounted for and, therefore, unadjusted densitometric measurements are biased. Different secretory capacities were corrected by expressing the densitometric measurements per unit secretory tissue. In this way, densitometric measurements from tissues with greater secretory capacity were adjusted upwards, and tissues with lower secretory capacities were adjusted downwards. Adjustments were calculated by the equation: adjusted arbitrary density units (ADU) = ADU/mass of tissue equivalents, where one tissue equivalent is the mass of tissue needed to synthesize and secrete 4 x 105 dpm of labeled macromolecules. Mass of tissue equivalents was obtained by dividing 4 x 105 dpm by incorporation rate (dpm nondialyzable macromolecules/mg of tissue) for individual tissue samples.

Hormone Assays

Concentrations of E2 and P4 in plasma were measured by RIAs previously validated in our laboratory (E2: [22]; P4: [23]). Intra- and interassay coefficients of variation were 15.5 and 12.4%, respectively, for E2 and, 6.8 and 8.1%, respectively, for P4.

Statistical Analysis

Data were analyzed by least-squares ANOVA using the General Linear Models of Statistical Analysis Systems (SAS) [24]. Concentrations of E2 and P4 in plasma and diameter of DF were analyzed by split-plot ANOVA. The mathematical model used treatment (FDF or PDF), cow (treatment), day, treatment by day, and error. Rate of incorporation of radioactivity into oviductal tissue and natural log of adjusted ADU measurement of proteins were calculated and analyzed by least-squares ANOVA. The mathematical model was treatment (FDF or PDF), cow (treatment), side (IPSI or CONTRA to the DF), region (INF, AMP, and IST), all higher-order interactions and error. Orthogonal contrasts for treatment (PDF vs. FDF), region (INF and AMP vs. IST and INF vs. AMP), and treatment-by-region interactions were used to compare means.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ultrasonography and Hormone Measurements

Size of DF was analyzed in two phases during the treatment period: from Day 5 to Day 9 (period before injection of GnRH) and from Day 10 until Day 16 (Fig. 2). Both FDF and PDF cows had similar sizes of DF from Day 5 to Day 9. However, a significant (p < 0.01) treatment-by-experimental-day interaction was detected from Day 10 to Day 16. All cows with FDF ovulated the first-wave DF, and a newly recruited DF was detected on Day 11 which reached 12 mm by Day 16. In contrast, the first-wave DF of the PDF group was sustained and reached a size of 22 mm by Day 16.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. Least-squares means (± SEM) of diameter of the DF of cows bearing an FDF (treated with GnRH on Day 9) or PDF (not treated with GnRH on Day 9) during the treatment period. Treatments with PGF2{alpha}, CIDR, and GnRH are indicated. Day 0 equals day of estrus at beginning of treatment period.

Concentrations of E2 (Fig. 3a) and P4 (Fig. 3b) were analyzed between experimental Day 7 (day of PGF2{alpha} injections) and Day 18 or 19. There was a significant (p < 0.01) treatment-by-experimental-day interaction for both E2 and P4 concentrations in plasma. After GnRH injection on Day 9, E2 concentrations decreased in plasma of FDF cows and remained between 5 and 10 pg/ml until Day 16 and increased to 22 pg/ml at Day 18 (estrus). For PDF cows, E2 remained at approximately 15 pg/ml from Day 9 until Day 19. After PGF2{alpha} injection on Day 7, P4 concentrations decreased for both groups between Day 7 and Day 11. After Day 11, P4 increased in association with development of a new CL in FDF cows (3 of 3) while concentrations of P4 remained low for PDF cows. After CIDR removal and PGF2{alpha} injection on Day 16, P4 concentrations decreased for FDF and PDF cows.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 3. Least-squares means (± SEM) of E2 (a) and P4 (b) concentrations in plasma of cows bearing an FDF (treated with GnRH on Day 9) or PDF (not treated with GnRH on Day 9) during the treatment period. Treatments with PGF2{alpha}, CIDR, and GnRH are indicated. Day 0 equals day of estrus at beginning of treatment period.

Incorporation Rate

Incorporation rate of radiolabel into protein can be used as a measure of the protein biosynthetic activity of tissues (i.e., amount of [3H]leucine incorporated into newly synthesized and secreted macromolecules). There was a significant (p < 0.05) treatment-by-region interaction (Fig. 4). The FDF increased incorporation rate of [3H]leucine into proteins in all oviductal regions (p < 0.01). However, stimulation was greatest in the INF. No side or side-by-treatment effects were detected (p > 0.1).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 4. Least-squares means (± SEM) of incorporation rates of [3H]leucine into INF, AMP, and IST of cows bearing an FDF (treated with GnRH on Day 9) or PDF (not treated with GnRH on Day 9) during the treatment period.

Fluorography and Densitometry

The pattern of proteins secreted by explants of INF, AMP, and IST, as resolved by two-dimensional SDS-PAGE, are shown in representative fluorographs in Figure 5. Proteins analyzed were designated P1 to P20 on the basis of their location in the fluorograph according to a clockwise pattern starting in the top left quadrant. Results of densitometric analyses of P1 to P20 are depicted in Tables 1 and 2. There was a trend for effect of treatment for P2 (p < 0.06) and P13 (p < 0.07). Interpretation of main effects of treatments on secretion of proteins indicates that the presence of FDF stimulated greater secretion of P2 and P13 than in tissues from PDF cows. There was a significant (p < 0.05) region effect for P1, P4–7, P12–17, and P20, and a tendency (p < 0.1) for P2, P3, P11, and P18. Effects of side were significant for P5 (p < 0.05) and P19 (p < 0.05) and approached significance for P8 (p < 0.1) and P15 (p < 0.06). Region-by-treatment interactions were significant for P7 and P14 (p < 0.02) and tended to be significant for P1 (p < 0.07). As suggested by significant treatment-by-region interactions, protein synthesis and secretion in response to treatments varied according to region. In the AMP, P7 was stimulated in PDF whereas P14 was stimulated in FDF cows. Protein 14 was present in the INF and absent in the IST regardless of treatment, but ampullary P14 was abolished by PDF. In the IST, synthesis of P1 was stimulated by PDF. Proteins with significant side-by-treatment interactions were P6 (p < 0.05), P9 (p < 0.05), and P11 (p < 0.03), while P5 (p < 0.06), P8 (p < 0.07), and P19 (p < 0.09) only approached significance. Treatment-by-side interactions indicate a differential response of IPSI and CONTRA sides to FDF compared to PDF. For FDF cows, abundance of P5, P6, P8, P9, and P11 was reduced in the IPSI compared with the CONTRA side. In contrast, abundance of these same proteins was similar across sides for PDF cows. Protein 19 was secreted in similar amounts at the IPSI side for both treatments. In the CONTRA side, however, PDF maintained, while FDF reduced, abundance of P19 compared to the IPSI side. Treatment-by-side-by-region interaction was significant for P19 (p < 0.01) and tended to be significant for P2 (p < 0.1) and P13 (p < 0.06).



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 5. Representative fluorographs of the 2D SDS-PAGE analysis of culture medium conditioned by explants of INF (a), AMP (b), and IST (c). Proteins analyzed by densitometry are indicated. Molecular weight standards are indicated (x10-3), and the pH gradient runs from left (pH 8) to right (pH 4).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Molecular masses (m)a and least-squares means and probability values of the logarithm of densitometric units of region by treatment interactions of selected proteins (P) on 2D gels.b


View this table:
[in this window]
[in a new window]
  		TABLE 2. Molecular masses (m)a and least-squares means and probability values of the logarithm of densitometric units of side by treatment interactions of selected proteins (P) on 2D gels.b

A summary of mean comparisons of effects of treatment, side, treatment-by-region, and treatment-by-side for individual spots is presented in Table 3.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Summary of means comparison of effects of treatment, treatment by region, side, and treatment by side interactions on the logarithm of densitometric units of selected proteins in 2D gels.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have shown that hormonal manipulations of animals altered both their follicular and luteal functions, which provided a model to study oviductal protein synthesis and secretion at estrus from distinctly different periestrus hormonal environments (Figs. 2 and 3). Distinctly different steroidal environments for cows bearing PDFs or FDFs specifically modulated biosynthetic activity, and protein synthesis and secretion from different functional regions of the oviduct. This altered pattern of protein synthesis and secretion in cows bearing PDFs may contribute to the lower fertility of this group of animals compared to cows ovulating FDFs [7]. Wehrman et al. [25] showed that normal embryos transferred 7 days after estrus to uteri of cows that ovulated a PDF had no difference in pregnancy rates compared to those of controls. This supports the concept that low fertility associated with PDF may be due to an inappropriate oviductal, not uterine, environment before Day 7, or an abnormal embryo [9, 10, 26].

Both ultrasonography and hormonal data indicated that the first-wave DF of the experimental period was ovulated after injection of GnRH agonist at Day 9 (FDF cows), whereas no injection of GnRH agonist (PDF cows) permitted sustained development of the first-wave DF. Continued growth of the first-wave DF occurred in the low-P4 environment supported by the CIDR in the PDF group. The PDF maintained high concentrations of estradiol in plasma. Cows of the FDF group ovulated the first-wave DF in response to GnRH, and the resulting CL secreted increasing amounts of P4 after Day 13. A newly selected FDF was detected by Day 11. Changes in E2 and P4 between PDF and FDF groups reflected the differences in CL and follicle dynamics also reported by Schmitt et al. [21]. The acute increase in E2 concentration in the FDF group was associated with development of a DF during the proestrous period after injection of PGF2{alpha} and withdrawal of the CIDR.

These endocrine environments induced distinctly different patterns of protein synthesis and secretion by all oviductal regions in cows with PDF vs. FDF. Studies in sheep [14], baboons [16], and pigs [17, 18, 27] indicate that specific oviductal proteins and specific mRNAs are regulated by endogenous steroids during the estrous cycle or early pregnancy and by exogenous steroids in ovariectomized animals. In the pig, a family of related glycoproteins (POSP 1–3), a basic and an acidic 100 000-Mr protein and a very acidic protein (75 000–85 000 Mr), are synthesized primarily by the AMP during proestrus, estrus, and metestrus (high E2, low P4) but not diestrus (low E2, high P4). Consistent with POSP protein synthesis and secretion, POSP mRNA expression is also estrogen-dependent and is significantly greater in the AMP on Day 0 and Day 1 of estrous cycle or pregnancy [28]. The cow oviduct produces a basic 97-kDa protein [29] similar to POSP, identified as P1 in this study. Protein 1 secretion reaches a maximum at estrus and decreases during the luteal phase of the estrous cycle. In agreement with Boice et al. [29], P1 was secreted by all oviductal regions in the present study. Other proteins, such as tissue inhibitor of metalloproteinase-1, found to be produced by the pig and cow oviduct (P20 in this study), was shown to be expressed optimally on Day 2 in the pig, when E2 and P4 were both low [30]. Further, E2 appeared to suppress synthesis and/or secretion of several protein complexes that were not identified [30]. Therefore, it was expected that the sustained high E2/low P4 milieu of PDF cows and the increasing E2 and P4 milieu of FDF cows would differentially modulate protein synthesis and secretion in the oviduct.

Analysis of the biosynthetic activity (incorporation rates) indicated that the presence of a PDF decreased the synthetic activity of oviductal tissues. However, the overall increased incorporation of label into synthesized and secreted macromolecules for FDF cows compared to PDF cows in all oviductal regions appears to be in variance from the similar abundance of specific proteins for both PDF and FDF cows (Tables 1 and 2). Several explanations for this dichotomy are possible. Higher E2 and lower P4 concentrations associated with presence of a PDF over an extended period of time apparently had a suppressive effect on overall biosynthetic activity of the different oviductal regions. Prolonged exposure to high levels of E2 in PDF cows may have caused down-regulation of E2 receptor, which would explain the suppression in biosynthetic activity of oviducts from PDF cows compared to FDF cows. However, synthesis and secretion of proteins that are actually inhibited by E2 could be stimulated as a result of down-regulation of E2 receptors. Studies in the hen [31], mouse [32], pig [33], and primates [34] indicate that absolute and relative amounts of receptors for E2 and P4 vary in the oviduct during the estrous cycle and pregnancy. Moreover, oviductal functions such as velocity of egg transport [32] and oviductal epithelial cell proliferation [34] also change in response to manipulations of the steroid environment and steroid binding to their receptors. Therefore, concentrations of circulating E2 and P4 for FDF and PDF cows may have differentially regulated numbers of E2 and P4 receptors in the oviduct, and consequently, expression of steroid-responsive proteins (as illustrated by changes in oviductal function). Alternative explanations for the discrepancy between incorporation rate and abundance of specific protein spots are 1) that in FDF cows, incorporation may have been greater in proteins of higher (> 97 kDa) or lower (< 20 kDa) molecular mass that were not resolved and would be undetectable in gel analyses; or 2) that FDF may have induced a higher turnover of proteins, and the resulting partially degraded proteins were not resolved by electrophoresis (i.e., molecular mass between 3.5 and 20 kDa).

Several studies in cattle suggest the importance of the oviductal region and protein milieu in reproductive processes. In studies with bulls [11], it has been demonstrated that culture medium conditioned by IST tissue at estrus capacitated more sperm than did medium conditioned by AMP. This increase was abolished by heating the conditioned medium and inactivating proteins before incubation with sperm. Staros and Killian [35] showed that four unidentified oviductal proteins and a P1-like protein from nonluteal oviductal fluid would associate with the zona pellucida, suggesting a modulation of sperm/egg binding or embryonic development by oviduct-derived proteins. In the present experiment, of 20 proteins analyzed by densitometry, P2 and P13 showed differences in synthesis between treatments. Both proteins were inhibited by PDF, suggesting an overall down-regulation. The strong effect of region for 16 of 20 spots measured suggested a biosynthetic gradient in which the secretion was greater or less for the IST depending upon the protein. Such a gradient has been reported in the pig and sheep [15, 28, 30, 36]. Moreover, DeSouza and Murray [36] reported differential secretion of a chitinase-like protein, similar to P1 in response to steroid treatments in sheep, while Buhi et al. [28] showed differential expression POSP mRNA among oviductal regions in pigs.

Treatment-by-region interactions indicate that the steroid milieu generated by PDF vs. FDF modulated synthesis and secretion of particular proteins differently, depending on the oviductal region. As an example, PDF abolished synthesis of P14 in the AMP, while P14 was absent in the IST and present in the INF regardless of the treatment. In contrast, P7 synthesis was induced by PDF in the AMP, although P7 was present in similar amounts for both treatments in the INF and IST. It is likely that optimal function of each region is achieved when the oviductal microenvironment includes the appropriate secretory proteins.

Treatment-by-side interactions indicated that the oviduct adjacent to the ovary bearing the DF responded differently depending upon follicular status (FDF vs. PDF). Ireland et al. [37] demonstrated that blood drainage from the ovary containing the DF contained a higher concentration of E2 than did that from the CONTRA ovary. Exposure to higher concentrations of E2 may therefore preferentially alter synthesis of selected proteins. In the present study, P5, P6, P8, P9, and P11 were reduced in the IPSI side compared to the CONTRA side in FDF cows, whereas for PDF cows, abundance was similar regardless of the side. This indicates that PDF overrode the side-dependent regulation of secretion of P5, P6, P8, P9, and P11 that occurs normally in FDF cows.

In summary, FDF and PDF regulation of protein synthesis and secretion in the oviduct is protein-, region-, and side-specific. This suggests that several mechanisms are involved in the complex regulation of oviduct function. Possible mechanisms include E2 and P4 receptor regulation; differential action of E2 and P4 (i.e., stimulatory vs. inhibitory) depending on protein, side and region; effects of autocrine and paracrine factors; cross-talk between E2- and P4-induced signal transduction and other intracellular effector systems.

In addition to steroids, it is possible that other effectors may control oviductal protein synthesis and secretion. A low-P4 environment elicited by a progestin-containing device in PDF cows is associated with higher LH pulse frequency compared to that in FDF cows [5, 6]. Derecka et al. [38] reported the presence of LH receptor mRNA in porcine oviduct tissue. Recently, LH receptor protein and mRNA transcripts were described in bovine oviductal epithelial cells [39]. Moreover, the authors of that study reported that hCG treatment of bovine oviductal epithelial cells in vitro induced time- and dose-dependent secretion of a 95-kDa oviductal glycoprotein. Therefore, it is possible that different patterns of LH release may directly affect the oviduct and modulate differential protein synthesis in FDF compared to PDF cows.

The present study identified a series of proteins in which synthesis and secretion are modulated differentially according to exposure of the oviduct to the in vivo steroid milieu. This indicates that the oviductal microenvironment is altered. Collectively, our findings add support to the concept that a less than optimal oviductal microenvironment may contribute to the low fertility of cows bearing a PDF. We propose that a combination of the effects of premature maturation of oocytes and inappropriate oviductal microenvironment is responsible for the decreased fertility observed in cows bearing PDF.


   ACKNOWLEDGMENTS
 
The staff of the Dairy Research Unit of the University of Florida for animal care, especially Dale Hissem, James Lindsey, and Mary Russell; Nina Nusbaum, Eddie Fredriksson, Monte Meyer, Dr. Eric Schmitt, Dr. Luzbel de la Sota, and Dr. Thais Diaz for help with management of cattle; Jesse Johnson and Idania Alvarez for laboratory expertise; Dr. Nancy Denslow and Dr. Lannett Edwards for help with densitometry; and Mary Ellen Hissem for help with preparation of the manuscript.


   FOOTNOTES
 
1 Supported by USDA-BARD Grant 94–34339–1212. Florida Agricultural Experiment Station Journal Series no. R-06619. Back

2 Correspondence: W.W. Thatcher, Department of Dairy and Poultry Sciences, University of Florida, Sheally Drive, Gainesville, FL 32611–0920. FAX: 352 392 5595; thatcher{at}dps.ufl.edu Back

3 Current address: Animal Science Research Center, University of Missouri, Columbia, MO 65211. Back

Accepted: February 19, 1999.

Received: November 11, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Thatcher WW, de la Sota RL, Schmitt EJ-P, Diaz TC, Badinga L, Simmen FA, Staples CR, Drost M. Control and management of ovarian follicles in cattle to optimize fertility. Reprod Fertil Dev 1996; 8:203–217.[CrossRef][Medline]
  2. Macmillan KL, Peterson AJ. A new intravaginal progesterone releasing device for cattle (CIDR-B) for oestrus synchronization, increasing pregnancy rates, and the treatment of post-partum anestrus. Anim Reprod Sci 1993; 33:1–9.
  3. Savio JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle in heifers. J Reprod Fertil 1988; 83:663–671.[Abstract]
  4. Sirois J, Fortune J. Ovarian follicular dynamics during the estrous cycle monitored by real time ultrasonography. Biol Reprod 1988; 39:308–317.[Abstract]
  5. Kinder JE, Kojima FN, Bergfeld EGM, Wehrman ME, Fike KE. Progestin and estrogen regulation of pulsatile LH release and development of persistent ovarian follicles in cattle. J Anim Sci 1996; 74:1424–1440.[Abstract]
  6. Savio JD, Thatcher WW, Badinga L, de la Sota RL, Wolfeson D. Regulation of dominant follicle turnover during the oestrous cycle in cows. J Reprod Fertil 1993; 97:197–203.[Abstract]
  7. Savio JD, Thatcher WW, Morris GR, Entwistle K, Drost M, Mattiacci MR. Effects of induction of low progesterone concentrations with a progesterone-releasing intravaginal device on follicular turnover and fertility in cattle. J Reprod Fertil 1993; 98:77–84.[Abstract]
  8. Cooperative Regional Research Project, NE-161. Relationship of fertility to patterns of ovarian follicular development and associated hormonal profiles in dairy cows and heifers. J Anim Sci 1996; 74:1943–1952.[Abstract]
  9. Ahmad N, Schrick FN, Butcher RL, Inskeep EK. Effects of persistent follicles on early embryonic losses in beef cows. Biol Reprod 1995; 52:1129–1135.[Abstract]
  10. Revah I, Butler WR. Prolonged dominance of follicles and reduced viability of bovine oocytes. J Reprod Fertil 1996; 106:39–47.[Abstract]
  11. Anderson SH, Killian GJ. Effect of macromolecules from oviductal conditioned medium on bovine sperm motion and capacitation. Biol Reprod 1994; 51:795–799.[Abstract]
  12. Boatman EB, Magnoni GE. Identification of a sperm penetration factor in the oviduct of the golden hamster. Biol Reprod 1995; 52:199–207.[Abstract]
  13. Gandolfi F, Brevini TAL, Richardson L, Brown CR, Moor RM. Characterization of proteins secreted by sheep oviductal epithelial cells and their function in embryonic development. Development 1989; 106:303–312.[Abstract]
  14. Buhi WC, Bazer FW, Alvarez IM, Mirando MA. In vitro synthesis of oviductal proteins associated with estrus and 17ß-estradiol-treated OVX ewes. Endocrinology 1991; 128:3086–3095.[Abstract]
  15. Murray MK. An estrogen-dependent glycoprotein is synthesized and released from the oviduct in a temporal- and region-specific manner during early pregnancy in the ewe. Biol Reprod 1993; 48:446–453.[Abstract]
  16. Verhage HG, Fazleabas AT. In vitro synthesis of estrogen-dependent proteins by baboon (Papio anubis) oviduct. Endocrinology 1988; 123:552–558.[Abstract]
  17. Buhi WC, Vallet JL, Bazer FW. De novo synthesis of polypeptides from cyclic and early pregnant porcine oviductal tissue in explant culture. J Exp Zool 1989; 252:79–88.[CrossRef][Medline]
  18. Buhi WC, Alvarez IM, Sudhipong V, Dones-Smith MM. Identification and characterization of the de novo-synthesized porcine oviductal secretory proteins. Biol Reprod 1990; 43:929–938.[Abstract]
  19. Van Cleef JK, Lucy MC, Wilcox CJ, Thatcher WW. Plasma and milk progesterone and plasma LH in ovariectomized lactating cows treated with new or used controlled internal drug release devices. Anim Reprod Sci 1992; 27:91–106.
  20. Macmillan KL, Taufa VK, Barnes DR, Day AM, Henry R. Detecting estrus in synchronized heifers using tailpaint and an aerosol raddle. Theriogenology 1988; 30:1099–1114.
  21. Schmitt EJ-P, Drost M, Diaz T, Roomes C, Thatcher WW. Effect of gonadotropin-releasing hormone agonist on follicle recruitment and pregnancy rate in cattle. J Anim Sci 1996; 74:154–161.[Abstract]
  22. Badinga L, Driancourt MA, Savio JD, Wolfenson D, Drost M, de la Sota RL, Thatcher WW. Endocrine and ovarian responses associated with the first-wave dominant follicle in cattle. Biol Reprod 1992; 47:871–883.[Abstract]
  23. Knickerbocker JJ, Thatcher WW, Bazer FW, Drost M, Barron DH, Fincher KB, Roberts RM. Proteins secreted by day-16 to-18 conceptuses extend corpus luteum function in cows. J Reprod Fertil 1986; 77:381–391.[Abstract]
  24. SAS. SAS/STAT User's guide (Release 6.03). Cary, NC: Statistical Analysis System Institute, Inc.; 1988.
  25. Wehrman ME, Fike KE, Melvin EJ, Kojima FN, Kinder JE. Development of a persistent ovarian follicle and associated elevated concentrations of 17ß-estradiol preceding ovulation does not alter the pregnancy rate after embryo transfer in cattle. Theriogenology 1997; 47:1413–1421.
  26. Mihm M, Currant N, Hytell P, Boland MP, Roche JF. Resumption of meiosis in cattle oocytes from pre-ovulatory follicles with a short and long duration of dominance. J Reprod Fertil Abstr Ser 1994; 13:14.
  27. Buhi WC, Alvarez IM, Pickard AR, McIntush EW, Kouba AJ, Ashworth CJ, Smith MF. Expression of tissue inhibitor of metalloproteinase-1 protein and messenger ribonucleic acid by the oviduct of cycling, early-pregnant, and OVX steroid-treated gilts. Biol Reprod 1997; 57:7–15.[Abstract]
  28. Buhi WC, Alvarez IM, Choi I, Cleaver BD, Simmen FA. Molecular cloning and characterization of an estrogen-dependent porcine oviductal secretory glycoprotein (POSP). Biol Reprod 1996; 55:1305–1314.[Abstract]
  29. Boice ML, Geisert RD, Blair RM, Verhage HG. Identification and characterization of bovine oviductal glycoproteins synthesized at estrus. Biol Reprod 1990; 43:457–465.[Abstract]
  30. Buhi WC, Ashworth CJ, Bazer FW, Alvarez IM. In vitro synthesis of oviductal secretory proteins by estrogen-treated ovariectomized gilts. J Exp Zool 1992; 262:426–435.[CrossRef][Medline]
  31. Kawashima M, Takahashi T, Kamiyoshi M, Tanaka K. Effects of progesterone, estrogen, and androgen on progesterone receptor binding in hen oviduct uterus (shell gland). Poult Sci 1996; 75:257–260.[Medline]
  32. Fuentealba B, Nieto M, Croxatto HB. Estrogen and progesterone receptors in the oviduct during egg transport in cyclic and pregnant rats. Biol Reprod 1988; 39:751–757.[Abstract]
  33. Stanchev P, Rodriguez-Martinez H, Edqvist LE, Eriksson H. Progesterone receptors in the pig oviduct during the oestrus cycle. J Steroid Biochem 1985; 22:115–120.[CrossRef][Medline]
  34. Slayden OD, Brenner RM. RU 486 action after estrogen priming in the endometrium and oviducts of rhesus monkeys (Macaca mulatta). J Clin Endocrinol Metab 1994; 78:440–448.[Abstract]
  35. Staros AL, Killian GJ. In vitro association of six oviductal fluid proteins with the bovine zona pellucida. J Reprod Fertil 1998; 112:131–137.[Abstract]
  36. DeSouza M, Murray MK. An estrogen-dependent secretory protein, which shares identity with chitinases, is expressed in a temporally and regionally specific manner in the sheep oviduct at the time of fertilization and embryo development. Endocrinology 1995; 136:2485–2496.[Abstract]
  37. Ireland JJ, Fogwell RL, Oxender WD, Ames K, Cowley JL. Production of estradiol by each ovary during the estrous cycle of cows. J Anim Sci 1994; 59:764–771.
  38. Derecka K, Pietila EM, Rajaniemi HJ, Ziecik AJ. Cycle dependent LH/hCG receptor gene expression in porcine non-gonadal reproductive tissues. J Physiol Pharmacol 1995; 46:77–85.[Medline]
  39. Sun T, Lei ZM, Rao CV. A novel regulation of the oviductal glycoprotein gene expression by luteinizing hormone in bovine tubal epithelial cells. Mol Cell Endocrinol 1997; 131:97–108.[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Binelli, M.
Right arrow Articles by Thatcher, W. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Binelli, M.
Right arrow Articles by Thatcher, W. W.
Agricola
Right arrow Articles by Binelli, M.
Right arrow Articles by Thatcher, W. W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS