Changes in the messenger RNA expressions of the endothelin-1 and angiotensin systems in mature follicles of the superovulated bovine ovary

The aim of the present study was to examine the messenger RNA expressions of the endothelin and angiotensin systems during the periovulatory phase in gonadotrophin releasing hormone (GnRH)-treated cows. Ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (control, before LH surge), 3-5 h after GnRH (during LH surge), 10 h after GnRH; 20 h after GnRH, 25 h after GnRH (peri-ovulation), and early corpus luteum (CL) (Days 2-3). Expression of mRNA was investigated using quantitative real-time PCR. The expression of angiotensin converting enzyme (ACE) mRNA significantly decreased immediately after onset of the LH surge and remained at low levels. The levels of angiotensin II receptor type 1 (AT1R) and type 2 (AT2R) expression during the periovulatory period significantly decreased compared with other periods. The concentration of angiotensin II in follicular fluid began to increase 10 h after GnRH treatment and further increased as ovulation approached. The level of ET-1 mRNA significantly decreased 10 h after GnRH treatment compared with the levels before GnRH treatment and those of the early CL period. The expression of ETR-A and ETR-B mRNA during the periovulatory period were lower than in other periods. The expression of ECE-1 mRNA began to decrease in the LH surge period and significantly decrease in the periovulatory period compared with other periods. These results suggest that the vasoactive peptides angiotensin and endothelin may be associated with final maturation of follicles.     

Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.     

Expression and localization of IGF family members in bovine antral follicles during final growth and in luteal tissue during different stages of estrous cycle and pregnancy

The objectives of the study were to monitor the detailed pattern for mRNA expression (RT-PCR and RPA) of IGFs, IGFR-1, IGFBPs, GHR and localization of protein (immunohistochemistry) for IGF-1 and IGFR-1 in bovine follicle classes during final maturation and different corpus luteum (CL) stages during estrous cycle and during pregnancy. A relative high expression of IGF-1 in theca interna (TI) was observed before selection (E<0.5ng/mL). In GC, mRNA expression increased after selection. In contrast, IGF-2 was mainly expressed in the TI. The IGFR-1 mRNA was present in the TI and GC with increasing levels during final development. The expression results were confirmed by localization of IGF-1 and IGFR-1 proteins in GC and TI. There is clear evidence for the local expression of IGFBPs in TI and GC compartment with clear regulatory differences. In CL, the highest mRNA expression of IGF-1, IGF-2 and IGFR-1 was observed during early luteal phase, followed by a decrease, and then by a tendency of an increase during the mid and late luteal phases of the cyclic CL. This level remained low during pregnancy. Intense immunostaining for IGFR-1 in CL was observed mainly in large luteal cells. Evidence for a mRNA for all six IGFBPs were obtained with distinct differences for BP-3, -4 and -5. In conclusion, this comprehensive study gives clear evidence for an important role of the IGFs and IGFBPs in bovine follicular development and CL function. The relative amounts of IGFBPs may ultimately determine ovarian IGF action.     

Influence of bovine growth hormone and growth hormone-releasing factor on messenger RNA abundance of lipoprotein lipase and stearoyl-CoA desaturase in the bovine mammary gland and adipose tissue

Our objective was to determine the influence of bovine growth hormone (bGH) and bovine growth hormone-releasing factor (bGRF) administration on the mRNA abundance of lipoprotein lipase (LpL) and stearoyl-CoA desaturase (SCD). Primiparous Holstein cows received bGH, bGRF, or no treatment from 118 to 181+/-1 d postpartum. We hypothesized that bGH and bGRF treatment would increase the mRNA abundance of both SCD and LpL in the mammary gland with a corresponding reduction in adipose tissue. Milk yield significantly increased but milk fat percentage did not change as a result of bGH or bGRF treatment. Short-, medium-, and long-chain fatty acid concentrations in milk were not affected by either bGH or bGRF treatments, with the exception of a modest, but significant, increase in C16:1 and C18:1 following bGH treatment. Analysis was conducted on the genes encoding LpL (E.C. 3.3.1.34), a key enzyme involved in the uptake of fatty acids into tissues, and SCD (E.C. 1.14.99.5), which is the enzyme responsible for introducing delta9 double bonds in fatty acids of 16 and 18 carbons in length. In adipose tissue, treatment with bGH and bGRF reduced the mRNA abundance of LpL to 14.6 and 25.7% respectively, of that observed for control animals. Similarly, these treatments reduced the SCD mRNA abundance to undetectable levels in adipose tissue. In mammary gland, bGH and bGRF had no significant impact on LpL mRNA abundance. Bovine GH did not significantly affect SCD mRNA abundance in the mammary gland, and bGRF reduced SCD mRNA abundance. From this study to examine the role of bGH and bGRF on the expression of the genes encoding these key lipogenic enzymes in cattle, we conclude that the increased substrate required for enhanced milk fatty acid yield may have been provided through redirection of nutrients to the mammary gland away from adipose tissue and through overall increased metabolism in the mammary gland.     

Matrix metalloproteinases 2 and 9 activity in bovine synovial fluids

Matrix metalloproteinases (MMPs) are important enzymes found in connective tissues and thought to be involved in cartilage degradation. They are detectable in bovine synovial fluid and may play a destructive role in bovine septic arthritis. The MMP gelatinase enzymes were detected by gelatin zymography using image analysis of the gels. The active gelatinase levels were determined by a gelatin degradation enzyme-linked immunosorbent assay (ELISA). Increased concentrations of MMP-9 activity were found in the synovial fluids of cows with septic arthritis (P < 0.001) in comparison with fluids from normal joints. Using the gelatin degradation ELISA the net active gelatinases were measured, and significant increases were found in gelatinase bioactivities in synovial fluids from septic joint disease cases (P < 0.001). Increased concentrations of MMP-2 activity were found in the synovial fluids of cows with aseptic arthritis, which appeared to be playing an important role in degradation of articular cartilage in joint disease. This finding required further investigation.     

Fish meal supplementation increases bovine plasma and luteal tissue omega-3 fatty acid composition

The objective of this experiment was to determine if dietary inclusion of fish meal would increase plasma and luteal tissue concentrations of eicosapentaenoic and docosahexaenoic acids. Seventeen nonlactating Angus cows (2 to 8 yr of age) were housed in individual pens and fed a corn silage-based diet for approximately 60 d. Diets were supplemented with fish meal at 5% DMI (a rich source of eicosapentaenoic acid and docosahexaenoic acid; n = 9 cows) or corn gluten meal at 6% DMI (n = 8 cows). Body weights and jugular blood samples were collected immediately before the initiation of supplementation and every 7 d thereafter for 56 d to monitor plasma n-3 fatty acid composition and BW. Estrous cycles were synchronized using 2 injections of PGF(2α) administered at 14-d intervals. The ovary bearing the corpus luteum was surgically removed at midcycle (between d 10 and 12) after estrus synchronization, which corresponded to approximately d 60 of supplementation. The ovary was transported to the laboratory, and approximately 1.5 g of luteal tissue was stored at -80°C until analyzed for n-3 fatty acid content. Initial and ending BW did not differ (P > 0.10) between cows supplemented with fish meal and those with corn gluten meal. Plasma eicosapentaenoic acid was greater (P < 0.05) beginning at d 7 of supplementation and docosahexaenoic was greater (P < 0.05) beginning at d 14 of supplementation for cows receiving fish meal. Luteal tissue collected from fish meal-supplemented cows had greater (P < 0.05) luteal n-3 fatty acids and reduced (P < 0.05) arachidonic acid and n-6 to n-3 ratio as compared with tissue obtained from cows supplemented with corn gluten meal. Our data show that fish meal supplementation increases luteal n-3 fatty acid content and reduces available arachidonic acid content, the precursor for PGF(2α). The increase in luteal n-3 fatty acids may reduce PGF(2α) intraluteal synthesis after breeding resulting in increased fertility in cattle.     

Effects of storage and passage of bovine luteal endothelial cells on endothelin-1 and prostaglandin F2alpha production

To establish a storage system for isolated bovine luteal endothelial cells (LECs), we investigated the basal and tumor necrosis factor (TNF) alpha-stimulated production of endothelin-1 (ET-1) and prostaglandin (PG) F2alpha in unfrozen and frozen-thawed LECs until passage 10. LECs were obtained from developing corpora lutea (CL; days 5-7 of the estrous cycle) using enzymatic digestion and magnetic beads coated with lectin BS-1. The LECs were frozen at -80 C or further cultured and/or passaged until passage 10 in DMEM/Ham's F-12 supplemented with 10% calf serum. The hormonal productions of unfrozen and frozen/thawed LECs were compared through passages 2-10. When both the unfrozen and frozen/thawed cells reached confluence, the culture medium was replaced with fresh medium containing 0.1% bovine serum albumin (BSA), and the cells were incubated with TNFalpha (50 ng/ml) for 12 h. The basal productions of ET-1 and PGF2alpha by the unfrozen and frozen/thawed LECs were similar at passage 2. The basal production of PGF2alpha by LECs was not altered by passage and storage at -80 C, whereas the basal production of ET-1 decreased from passage 2 and 3 to passage 4 in the unfrozen LECs and from passage 2 to passage 3 in the frozen/thawed LECs. However, production of ET-1 by the unfrozen and frozen/thawed LECs was similar between passages 4-10 and passages 3-10, respectively. Exposure of LECs to TNFalpha increased (P<0.05) ET-1 and PGF2alpha production by the unfrozen and frozen-thawed LECs in all passages examined. Thus, LECs obtained from developing CLs and stored until passage 10 can be used for study of the physiology of LECs in vitro.     

Sequential medium with GH and IGF‐1 improved in vitro development of bovine preantral follicles enclosed in ovarian tissue

The growth hormone (GH) and growth insulin‐like factor‐1 (IGF‐1) act directly upon the regulation and growth in the different phases of preantral follicles. Thus, it is necessary to define their sequentiality until the in vitro preovulatory development. Therefore, the study aimed to assess the effects of a sequential medium containing GH and/or IGF‐1 in the long‐duration in vitro culture of preantral ovarian follicles. Ovarian fragments were cultivated: first half (days 1–7), second half (days 7–14) or during 14 culture days. Treatments were identified as: αMEM+; GH → IGF‐1; IGF‐1 → GH and GH + IGF‐1. The culture was designed in 24‐well plates, in an incubator at 37°C and 5% CO₂. The parameters of normality, viability, follicles (primordial/in developing) and follicle diameter were evaluated. In addition, the ultrastructure was confirmed with electron transmission microscopy. The results showed that the culture treated with GH → IGF‐1 kept the follicular normality and the viability until the 14th day of culture and increased both in the follicular development until 7th day and in the follicular diameter until 14th day, when compared to the control. The treatments IGF‐1 → GH and GH + IGF‐1 were not effective in the developing and follicular diameter after 7 days of culture, and also reduced the percentage of viability. It is concluded that the bovine preantral follicles cultured in the sequential medium treated with GH → IGF‐1 improved the follicular development until the first half of the culture and kept these parameters with normality, viability and ultrastructure until the second half of the in vitro culture.     

Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-1beta-stimulated matrix metalloproteinases 1, 3, and 13 and tissue inhibitor of matrix metalloproteinase 1 expression in equine chondrocyte cultures

OBJECTIVE: To determine the effects of prostaglandin E2 (PGE2) on recombinant equine interleukin (IL)-1beta-stimulated expression of matrix metalloproteinases (MMP 1, MMP 3, MMP 13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP 1) in vitro. SAMPLE POPULATION: Cultured equine chondrocytes. PROCEDURE: Stationary monolayers of first-passage chondrocytes were exposed to graduated concentrations of PGE2 with or without a subsaturating dose (50 pg/ml) of recombinant equine IL-1beta (reIL-1beta) to induce expression of MMP 1, MMP 3, MMP 13, and TIMP 1, followed by RNA isolation and northern blotting. In subsequent experiments, gene expression was similarly quantified from mRNA isolated from cultures pretreated with phenylbutazone to quench endogenous PGE2 synthesis, followed by exposure to reIL-1beta and exogenous PGE2 (5 mg/ml) with appropriate controls. RESULTS: Exogenous PGE2 (10 mg/ml) significantly reduced reIL-1beta-induced expression of MMP 1, MMP 3, MMP 13, and TIMP 1. Abrogation of cytokine induction with this dose of PGE2 was comparable to that for dexamethasone (10(-5) M) control. Similarly, pretreatment with phenylbutazone, followed by exposure to relL-1beta and PGE2 (5 mg/ml), was associated with a reduced expression of the genes of interest, an effect that was significant for MMP 1, MMP 13, and TIMP 1. CONCLUSIONS AND CLINICAL RELEVANCE: The MMP and TIMP 1 are important mediators in the pathophysiologic events in osteoarthritis. The potential for physiologically relevant regulation of expression of these genes by PGE2 is a consideration in the use of drugs that inhibit prostanoid synthesis in the treatment of equine arthropathies.     

Expression of fibroblast growth factor 1 (FGF1) and FGF7 in mature follicles during the periovulatory period after GnRH in the cow

The aim of this study was to evaluate the expression pattern of mRNA for fibroblast growth factor 1 (FGF1), FGF7, and their receptor variants (FGFR2IIIb) in time-defined follicle classes before LH surge, between LH surge and ovulation, and in the early corpus luteum (CL) in the cow. The ovaries were collected by transvaginal ovariectomy (n=5 cows/group), and the follicles (n=5, one follicle/cow) were classified into the following groups: before GnRH administration (before LH surge); 3-5 h after GnRH (during LH surge); 10 h after GnRH; 20 h after GnRH; 25 h after GnRH (periovulation), and early CL (Days 2-3). The mRNA expression was analyzed by quantitative real-time PCR (RotorGene 3000). The mRNA expression of FGF1 showed no significant differences in the follicle groups examined, but increased significantly at the early CL phase. A transient increase in FGF7 mRNA expression was observed 3-5 h after GnRH and again in the early CL phase. In contrast, the expression of FGFR2IIIb was constant throughout the period from the final growth of the follicle to early CL formation. The results of this study suggest that FGF1 and FGF7 may be involved differently in the process of follicle maturation and CL formation, which is strongly dependent on angiogenesis.     

Influence of polychlorinated biphenyls on the secretion of oxytocin from bovine luteal cells and from granulosa cells obtained from the follicles of different size

Effect of polychlorinated biphenyles (PCBs) on viability and secretory function of luteal and granulosa cells from mature cows was studied. Luteal cells from corpora lutea of different developmental stages and granulosa cells from follicles of >1 cm< in diameter were used. Neither individual congeners (PCB-126, -77, -153) nor mixture of PCBs Aroclor Ar) 1248 at the dose of 1, 10 or 100 ng/ml affected the viability of cells (P>0.05) compared to control after 72 h of incubation. PCBs markedly increased (P<0.05-0.001) oxytocin (OT) secretion from granulosa cells. This effect was the most evident when granulosa cells from follicles <1 cm diameter was treated with PCB-77 which is assumed to stimulate both arylhydrocarbon receptor (AhR) and estradiol (E2) receptor. Even the lowest dose of this compound (1 ng/ml) outranged the effect produced by cortisol (10(-5)M) used as positive control. There was marked effect (P<0.05-0.001) of PCBs on luteal cells from days 6-15 of the estrous cycle. However, influence of PCBs on OT secretion from luteal cells on day 1-5 and 16-18 of the estrous cycle was less evident. Again, PCB-77 was the most efficient stimulator of OT secretion. While the lowest effect was found after treatment of cells with PCB-126 which has dioxin-like properties. It can be assumed that diverse effect of PCBs on female reproduction largely results from the influence of these compounds on ovarian OT secretion. Since both synthesis and secretion of ovarian OT in bovine do not markedly depend on estradiol, some alternative cellular pathways of PCBs on ovary function are suggested.     

Effects of melatonin on morphology and development of primordial follicles during in vitro culture of bovine ovarian tissue

This study aims to investigate the effect of melatonin on activation, growth and morphology of bovine primordial follicles, as well as on stromal cells density in ovarian tissues after in vitro culture. Ovarian fragments were cultured in α‐MEM⁺ alone or supplemented with melatonin (250, 500, 1,000 or 2,000 pM) for a period of six days. Non‐cultured and cultured tissues were processed for histological analysis; according to developmental stages, follicles were classified as primordial or growing follicles. These follicles were further classified as morphologically normal or degenerated. Ovarian stromal cell density was also evaluated. The percentages of primordial and developing follicles, as well as those classified of normal follicles, were compared by Fisher's exact test, and the differences were considered significant when p < .05. The results showed that the presence of 1,000 and 2,000 pM melatonin in culture medium promoted a reduction in the percentage of primordial follicles and an increase in the percentage of development follicles, when compared to follicles cultured in control medium. On the other hand, the presence of 250 or 500 pM melatonin did not show a significant effect on the percentage of primordial and developing follicles. Besides that, the presence of 500, 1,000 and 2,000 pM melatonin maintained the percentage of normal follicles similar to those seen uncultured control. Moreover, tissues cultured in presence of 1,000 pM melatonin showed a higher percentage of normal follicles when compared to follicles cultured in the presence of 250 pM melatonin. It was observed a similar profile of stromal density in both uncultured tissues and those cultured in vitro in the presence of melatonin. In conclusion, melatonin (1,000 and 2,000 pM) promotes bovine primordial follicles activation and maintains the stromal cell density during in vitro culture of ovarian cortical tissue.     

Effects of tissue inhibitor of metalloproteinases on canine chondrocytes cultured in vitro with tumor necrosis factor-alpha

OBJECTIVE: To elucidate tissue inhibitor of metalloproteinase (TIMP)-mediated effects on chondrocytes. SAMPLE POPULATION: Articular cartilage from humeral heads of 6 dogs. PROCEDURE: Chondrocytes from harvested specimens were cultured in 3-dimensional (3-D) agarose at 10(6) cells/mL. We prepared 3-D constructs exposed to only tumor necrosis factor (TNF)-alpha (50 ng/mL). Recombinant human TIMP-1 (255nM), -2 (285nM), or -3 (250nM) was added to liquid media bathing 3-D constructs cultured with TNF-alpha. Chondrocytes cultured without TIMP or TNF-alpha served as control samples. Samples of liquid media were collected on days 6, 9, 15, and 21 of culture for evaluation of glycosaminoglycan (GAG) and nitric oxide concentrations. The 3-D constructs were collected on days 9, 15, and 21 for evaluation of GAG, hydroxyproline (HP), and DNA contents. RESULTS: GAG content in control samples increased significantly during the study, whereas GAG content in 3-D constructs cultured with TNF-alpha or TNF-alpha plus TIMP did not increase. On day 9, GAG release from 3-D constructs cultured with TNF-alpha was significantly higher than that in other constructs. The HP content in control samples increased during the study and was significantly higher than that in all other constructs on day 21. Concentrations of nitric oxide were significantly lower in control samples on day 6, compared with concentrations for all other constructs. CONCLUSIONS AND CLINICAL RELEVANCE: Addition of TIMPs did not counteract suppression of GAG and HP accumulation in 3-D constructs exposed to TNF-alpha. Apparently, adverse effects on chondrocytes exposed to TNF-alpha cannot be prevented by addition of TIMP alone.     

Immunohistochemical localization of IgG1 and IgG2 in prepartum and lactating bovine mammary tissue

The differential distributions of IgG1 and IgG2 were determined in prepartum and lactating bovine mammary tissue by indirect immunofluorescence. IgG1 was found predominately within the alveolar epithelial cells and lumens of prepartum tissue whereas IgG2 was largely confined to the stromal area surrounding the alveoli. Both IgG subclasses were confined predominately to the stroma in lactating tissue. Few IgG containing stromal cells were readily distinguished in any of the mammary tissue used in this study.     

Identification of soluble forms of vascular endothelial growth factor receptors, sVEGFR-1 and sVEGFR-2, in bovine dominant follicles

The development of dominant follicles requires the parallel growth of a vascular network, regulated by VEGF and its receptors VEGFR-1 and VEGFR-2. Here, we demonstrate the presence of mRNA for the soluble forms of VEGFR-1 and VEGFR-2 by RT-PCR and the respective proteins by Western blot, in bovine dominant follicles. The 3' end of the mRNA coding region and the deduced C-terminal amino acid sequence of the bovine VEGFR soluble forms were similar to those previously described in human and mice. The relative abundance of sVEGFR-1 was higher in dominant follicles of day 4, decreasing on day 6 and further on day 9 of the cycle. In contrast, sVEGFR-2 expression was low on day 4 follicles and increased as the cycle advanced, becoming greater on day 9. The changes of sVEGFR-1 and sVEGFR-2 with the age of the bovine dominant follicle indicate a physiological role in its growth and atresia.     

Estradiol, progesterone and cyclic nucleotides in the fluid of persistent follicles and luteal phase follicles in the estrus cycle in cows]

The objective of the present study was to compare the concentrations of 17 beta-estradiol, progesterone, cyclic adenosine monophosphate and cyclic quinosine monophosphate in the largest follicles of cows that persist for seven days after insemination following the preceding synchronization of oestrus and superovulation and in follicles of the luteal phase of cycle (5th-10th days). Animals included in the experiment were selected on the basis of rectal examination. Synchronization of oestrus was achieved in 24 crossbreds of Slovak Pied x Lowland Black Pied breeds (SS x Nc) using two doses of cloprostenol of Czechoslovak provenience Oestrophan Spofa, 500 micrograms in each, within 11 days. Serum gonadotrophin at the amount of 2500 I. U. was administered forty-eight hours before administration of the second dose PGF2 alpha. Experimental animals were inseminated after 72 hours. On the 7th day after mating the cows were killed at a slaughterhouse. Evaluated were only the ovaries of the 14 cows in which the persistent large follicles occurred. Ovaries of the 13 control cows in the luteal phase between the 5th-10th days were obtained at the slaughterhouse by the method after Ireland et al. (1980). Correct determination of the phase of sexual cycle was substantiated by determination of progesterone concentrations in blood serum. Follicular fluid was obtained from the largest follicles by aspiration and centrifuged in a cooled centrifuge at 3000 G. The concentrations of 17 beta-estradiol and progesterone in follicular fluid were determined using kits from URVJT at Kosice, designated RIA-test-ESTRA (SI-125-9) or RIA-test-Prog (SI-125-6).2+ persistent follicles (9.15 +/- 5.47 nmol.l-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of messenger ribonucleic acid encoding for steroidogenic acute regulatory protein and enzymes, and luteinizing hormone receptor during the spring transitional season in equine follicles

The period of spring transition, from the anovulatory to the ovulatory season, is characterized in many mares by cyclical growth and regression of large dominant follicles. These follicles produce only low concentrations of estradiol and it is thought that acquisition of steroidogenic competence by large follicles during spring transition is prerequisite in stimulating LH prior to first ovulation. In situ hybridization was used to localize and quantify expression of factors that play a key role in follicular steroidogenesis: StAR, P450scc (CYP11A1), P450c17 (CYP17), P450arom (CYP19), and LH receptor (LHr). One ovary was obtained from mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle (defined as the transitional follicle), and the remaining ovary was removed at the third estrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter (defined as the preovulatory follicle). Messenger RNAs encoding StAR, CYP11A1, and CYP17 were detected only in theca cells and CYP19 mRNA was confined to the granulosa layer. There was significantly lower expression of mRNAs for the steroidogenic enzymes, StAR (P<0.001) and LHr (P<0.05) in transitional follicles than in preovulatory follicles. In conclusion, large equine follicles during spring transition have low levels of mRNA encoding steroidogenic enzymes, StAR and LHr which will contribute to the steroidogenic incompetence of dominant follicles during spring transition and their subsequent regression.     

Prostaglandin F2alpha (PGF2alpha) independent and dependent regulation of the bovine luteal endothelin system

We have examined the genes of the endothelin system that are targets for regulation by prostaglandin F2alpha (PGF2alpha). The effects of a luteolytic dose of PGF2alpha ) on the mRNA encoding endothelin converting enzyme-1 (ECE-1), pre-pro endothelin-1 (pp ET-1) and the ET receptors ETA, ETB, in bovine corpus luteum (CL) during the early (days 1 and 4), mid (day 10) or late (day 17) luteal phases were examined. The effect of the PGF(2alpha) treatment on ECE-1 protein, Big ET-1 and the biologically active mature ET-1 peptide were also examined. Most importantly, the direct ECE-1 activity was determined. Before day 10 of the cycle, in a PGF2alpha-independent manner, the amounts of mRNA encoding ET-1, ECE-1, ETA, and ETB were increased steadily from day 1. After day 10 of the cycle, expression of mRNA encoding pp ET-1 and ETA acquired responsiveness to exogenous PGF2alpha and both genes were up-regulated by the PGF2alpha treatment. This effect of PGF2alpha was also detected for the proteins corresponding to the mature ET-1. The enzymatic activity of ECE-1 remained unchanged throughout the lifespan of the CL in spite of the detected changes in mRNA and protein. The results suggest that the luteal endothelin system is regulated in a PGF2alpha-independent and -dependent manner. Importantly, an alteration in luteal ET-1 availability is most likely achieved by modulating the expression of mRNA encoding pp ET-1 and not by the amount or activity of ECE-1. This interpretation is supported by the observation that the activity of ECE-1 remained unchanged throughout the ovarian cycle. The combined effects of greater ET-1 availability and gene expression encoding the ETA receptor in the late luteal phase could render the CL, at this developmental stage, more sensitive or responsive to ET-1. If the luteal tissue is responsive to the available ET-1 during the early phase of the ovarian cycle, an additional role for ET-1 should be considered beyond mediating the luteolytic actions of PGF2alpha. Agents blocking the actions of ET-1 might be the best approach to interfere with the luteal ET system and test its physiological role(s) in vivo.