Measurement of redox potential and steroid concentrations in the follicular fluid of growing and regressing follicles of mares

The aim of this study was to prove if oxidation-reduction levels in the follicular fluid were new functional indices of follicular health and whether there was a high level of accordance with endocrinological parameters and with the growth stage as detected by ultrasound monitoring of individual follicles during the oestrous cycle in mares. Follicles were classified as growing and regressing follicles using ultrasonography. Altogether 48 follicles with a diameter from 20 to 56 mm were aspirated by transvaginal ultrasound guided follicular aspiration. Follicular concentration of oestradiol and progesterone in relation to the diameter of growing follicles showed correlations of r = 0.64 and r = 0.57, respectively. The redox potential derived index D2 varied from -448 to +431 in the collected fluids of the follicles. The accordance of the judgement of all follicles using both complexes of methods - endocrinological and ultrasonographic parameters vs. analysis of oxidation and reduction levels - reached 72.5%. This finding has shown that parameters of redox reactions do not correlate closely with the stage of follicular growth or regression as determined by in vivo scanning of ovaries or by assessment of follicular steroid concentrations. However, the measurement of redox potentials offers an opportunity to examine the whole process of metabolism in follicular cells and to forecast impairments of cellular performances. Changes of redox parameters in growing follicles enable an earlier prediction of their further development. The data demonstrate that growing and regressing follicles do not represent nonatretic, early atretic and atretic follicles, respectively.     

Effect of exogenous ovarian steroids on the uterine luminal prostaglandins in ovariectomised mares with experimental endometritis

Prostaglandins (PGs) F and E2 were measured in lavage fluid from the uterus of ovariectomised mares after experimental induction of uterine inflammation. Treatment with progesterone alone, or progesterone followed by oestradiol, significantly increased the concentrations of these PGs in the lavage compared with mares treated with oestradiol or control mares. Ovarian steroids, therefore, influenced uterine PG synthesis in response to an inflammatory stimulus. To determine whether the uterine lavage procedure might contribute to the concentrations of prostaglandins in the lavage, the procedure was also performed on six intact mares. With the exception of washings obtained at luteolysis, uterine concentrations of PGF (measured as the plasma metabolite 15-keto-13,14-dihydro PGF2 alpha) had returned to prewashing levels within 30 minutes of the start of uterine lavage. Lavage was therefore unlikely to have influenced the concentrations of prostaglandins in the lavage fluid.     

Differential miRNA expression between equine ovulatory and anovulatory follicles

Relatively little is known about the physiological roles of microRNAs (miRNAs) during follicular development. Previous evidence from in vitro studies suggests specific roles for a subset of miRNAs, including miR-21, miR-23a, miR-145, miR-503, miR-224, miR-383, miR-378, miR-132, and miR-212, in regulating ovarian follicle development. The objective of this study was to gain insight on the involvement of these miRNAs during follicle maturation. Follicular fluid was aspirated from dominant follicles (>32 mm) during the ovulatory season (July to October) and the anovulatory season (January to March) in each of 5 mares, and the levels of steroids, IGF1, and miRNAs were analyzed by immunoassays and quantitative PCR. Levels of progesterone, testosterone, and IGF1 were lower (P ≤ 0.05) in anovulatory than in ovulatory follicles. Relative to ovulatory follicles, anovulatory follicles had higher (P < 0.05) mean levels of miR-21, miR-23b, miR-378, and miR-202 and tended to have higher (P = 0.06) levels of miR-145. Levels of miR-224 and miR-383 could not be detected in follicular fluid. These novel results indicate a physiological association between increases in follicular miRNA levels and seasonal anovulation in mares; further studies should elucidate the precise involvement of miR-21, miR-23b, miR-145, miR-378, and miR-202 in follicle maturation in the mare.     

Large variation in steroid concentrations and insulin-like growth factor binding proteins exists among individual small antral follicles collected from within cows at random stages of the estrous cycle

Variation in the biochemical status of individual small (< or = 5 mm diameter) antral follicles within the ovaries of a cow at any given time likely influences the capacity for undergoing recruitment, selection, and establishing dominance. The objectives of this study were to provide insight into the magnitude of variation in follicular fluid concentrations of steroids and activities of IGFBP that exists among individual small antral follicles within and between cows, and to determine the relationships between follicular fluid IGFBP and steroid concentrations in these follicles. A total of 108 small antral follicles were collected from 6 cows at random stages of the estrous cycle, with 10 to 26 follicles/cow. Concentrations of steroids (ng/mL of follicular fluid) in the overall population of follicles ranged from 0.1 (lowest detectable limit) to 51 for estradiol (E2), 4 to 1,149 for progesterone (P4), and 5 to 504 for androstenedione (A4). Concentrations of E2 and A4 were associated positively (r = 0.2; P < 0.02), but E2 (r = -0.4) and A4 (r = -0.4) were associated negatively, with P4. The proportion of variation in steroid concentrations accounted for by differences among animals (P < 0.05) was small for E2 (12%), moderate for P4 (43%), and greatest for A4 (74%). Least differences between minimum and maximum concentrations of steroids observed in follicles from within a cow were 21-, 5.5-, and 3.5-fold for E2, P4, and A4, respectively, whereas the greatest differences between minimum and maximum concentrations were 505-, 108-, and 26-fold for E2, P4, and A4, respectively. Ranges of IGFBP concentrations (arbitrary densitometer units) detected in fluid from a sub-sample of 43 follicles were 1.18 to 4.50 for IGFBP-3, 0.54 to 4.68 for IGFBP-2, 0.07 to 2.56 for IGFBP-4, and 0.01 to 6.71 for IGFBP-5. Concentrations of E2 were correlated negatively with each IGFBP (r = -0.4 to -0.8; P < 0.05) except IGFBP-3. In contrast, concentrations of A4 were correlated positively with IGFBP-3 (r = 0.4; P < 0.05) but were not correlated with other IGFBP. Concentrations of P4 were correlated positively (r > 0.4; P < 0.05) with IGFBP-4 and -5. The results indicate that steroid concentrations and IGFBP activities vary substantially among small antral follicles collected from within and among individual animals and that increasing production of E2, the hallmark of a developing follicle, was associated with reduced activity of all IGFBP except IGFBP-3, thereby implicating these IGFBP in the regulation of follicular recruitment.     

Effects of LH and PGF2alpha in equine dominant follicles observed by MDS

Microdialysis System (MDS) is a novel technique used for investigation of molecule secretion between different cell populations. Local hormonal secretion at follicular wall has been still unclear. This MDS study was used to determine progesterone (P4), androstenedione (A4), estradiol-17beta (E2) and Prostaglandin F2alpha (PGF2alpha) release in mare pre-ovulatory follicles. Follicles larger than 30 mm were isolated from the ovary and follicular fluid aspirated for hormone assay. Follicular fluid collected from small, middle and large follicles were analyzed by EIA. The concentrations of P4 and PGF2alpha were similar among the different sizes of follicles. The release of A4 was observed in middle and large follicles. E2 concentration was observed in middle follicles and was higher in large follicles compared with middle follicles. Follicular wall was cut and incubated for MDS and when LH was infused, there was an increase in P4 and A4 release. PGF2alpha release was considerably high after LH infusion compared to the control group. Infusion of PGF2alpha increased P4 and A4 release but there was no change in E2 release. This results suggest that in pre-ovulatory follicles, LH stimulates theca interna cells and also PGF2alpha seemed to have a mediator role to induce steroid hormone production and luteinization of follicular cells. The nature of the mechanisms involved in selection of large follicles is still a perplexing research problem in reproduction.     

Control of ovarian follicles activity in the ewe.

During the ovine estrous cycles, three waves of follicular growth, closely associated with the FSH secretion pattern, were observed. The parameters of these follicular waves and the ability of follicles to produce steroids in vitro were studied in various conditions. In vivo, the follicular events were similar between the breeding season and the anestrus, except for the lack of ovulation; but at the end of the breeding season and in anestrus, the follicles lose a big part of their aromatization ability. In ewes carrying the Booroola fecundity gene or Cambridge fecundity gene, the reduction in follicular atresia seems to be one of the main follicular features implicated in the control of high ovulation rate. In vitro, the most relevant difference is an early acquisition of estrogen production ability of small follicles in Booroola fecundity gene barring ewes. Fluoro-gestone-acetate (FGA) pessaries reduced the number of growing follicles; despite this effect disappearing after the sponge withdrawal, the ovulation rate is significantly reduced. But an equine chorionic gonadotrophin (eCG) treatment restores the ovulation rate (OR) by reducing the atresia rate of pre-ovulatory follicles. In similar conditions, a pretreatment of the ewes with melatonin again reduced the atresia rate of large follicles and resulted in an increased ovulation rate. In vitro, FGA blocked aromatization ability, and melatonin inhibited both androstenedione and estradiol production, but a further treatment with eCG partly restores the steroid secretion. Immunization against androstenedione leads to a higher OR, owning to a reduced atresia of large follicles. Daily growth hormone injections for a hole cycle resulted in an increased follicular population and ovulation rate, while FSH plasma levels decreased and the follicle sensitivity to gonadotrophins was reduced.     

Comparison of leptin levels in serum and follicular fluid during the oestrous cycle in cows

Leptin is mainly synthesised in white adipose tissue. Besides its effects on body weight and metabolic homeostasis, leptin also has effects on puberty, sexual maturation and reproduction. In this study the relationship between leptin, IGF-1, oestradiol (E2) and progesterone levels were investigated in serum and follicular fluid from cows. This study included 72 healthy, Brown Swiss cows aged 4-5 years. Samples from the jugular vein and follicular fluids were collected. Phases of the oestrus cycle of cows were classified according to their serum progesterone levels (< 3.18 nmol/l, follicular phase and the others as luteal phase). Follicles were grouped as large (> or = 8 mm) or small (< 8 mm). Leptin, IGF-1, oestradiol and progesterone levels were measured from serum and follicular fluid. Leptin concentrations were found to be significantly higher in luteal-phase follicular fluid of small follicles (P < 0.05). These were classified as atretic follicles. There was a positive correlation between serum and follicular fluid leptin levels in the luteal phase. Serum leptin was found to have a positive correlation with follicular fluid progesterone level (P = 0.01) in the preovulatory follicles. The present study shows that there is a relationship between the concentration of leptin in follicular fluid and atresia in small follicles.     

Effect of Suppression of FSH with a GnRH Antagonist (Acyline) Before and During Follicle Deviation in the Mare

A GnRH antagonist (Acyline) was used to study the role of FSH in early development of a follicular wave in 61 mares. In Experiment 1, a single dose of 3 mg per mare, compared with 0 and 1 mg, suppressed both the FSH and follicle responses to exogenous GnRH. In Experiment 2, high concentrations of FSH were induced by two successive ablations of all follicles ≥ 6 mm on days 10 and 13 (day 0 = ovulation). A single treatment with Acyline resulted in significantly greater suppression of plasma concentrations of FSH than a single treatment with charcoal-extracted follicular fluid (source of inhibin) or oestradiol. Suppression of FSH was not significantly different between the group treated with Acyline alone and a group treated with a combination of Acyline, inhibin and oestradiol. In Experiment 3, all follicles were ablated on day 10 to induce an FSH surge and a new follicular wave. Acyline treatment on day 10 resulted in an immediate decrease in FSH, without a significant effect on day of emergence of a new wave or growth of follicles from 7 to 11 mm on days 11–13. Treatment on day 15, a day before expected follicle deviation and after the peak of the wave-stimulating FSH surge, resulted in an immediate decrease in FSH and cessation of follicle growth. Results indicated that growth of follicles for about 2 days after wave emergence was independent of FSH. In contrast, during the decline in the wave-stimulating FSH surge and before follicle deviation, growth of follicles was dependent on FSH.     

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)     

Passive immunization of cyclic mares against androgen: gonadotropin and progesterone concentrations and estrous characteristics

Antiserum generated in a horse against testosterone conjugated to bovine serum albumin (BSA) was administered to six lighthorse mares (androgen-immunized mares) 1 to 3 d before a prostaglandin-induced estrus and twice again at 2-d intervals. Six control mares were administered antiserum generated against BSA on the same schedule. Relative to testosterone, cross-reactivities of other steroids with the testosterone antiserum were (%): dihydrotestosterone, 52; 5 alpha-androstane-3 alpha,17 beta-diol, 8.6; androst-4-ene-3,17-dione, 1.2; and all others tested less than .1. Tritiated testosterone binding in plasma increased (P less than .01) in androgen-immunized mares within 1 h and remained elevated (P less than .01) relative to controls for greater than 21 d. There was no effect (P greater than .10) of passive immunization against androgen on interval to estrus after prostaglandin injection, duration of estrus, ovarian volume, number of palpable follicles or follicular volume during estrus. In contrast, concentrations of luteinizing hormone (LH) were higher (P less than .05) in androgen-immunized mares than in control mares during estrus and early diestrus. Concentrations of follicle stimulating hormone (FSH) and progesterone at those times were not affected (P greater than .10). From these data, we conclude that androgens in the mare during estrus may be involved with the regulation of LH secretion. In contrast, no involvement with FSH secretion was apparent under these short-term conditions.     

Dynamics of Follicular Fluid in One‐humped Camel (Camelus dromedarius)

In the present study, ovarian follicular fluid and serum biochemical, hormonal, electrolytes and amino acids profiles in female dromedary camel (Camelus dromedarius), were investigated. Fluid from small (2–6 mm) and large follicles (7–20 mm) and blood samples were collected from 25 clinically healthy adult female camels. The concentrations of glucose, cholesterol, triglycerides, high‐density lipoproteins, urea, total proteins, albumin, globulin, fibrinogen, alanine aminotransferase, aspartate aminotransferase and tri‐iodothyronine were lower (p ≤ 0.05) in large follicles when compared with the small follicles. However, the concentrations of low‐density lipoproteins, uric acid, creatinine, alkaline phosphatase and acid phosphatase in small and large follicles did not differ. The concentrations of oestradiol 17‐β and progesterone were higher (p ≤ 0.05) in large follicles. The serum concentrations of these hormones were many folds lower (p ≤ 0.05) than those of follicular fluid. Among electrolytes, the concentration of phosphorus was higher (p ≤ 0.05) in the large follicles, while that of potassium and chloride were lower (p ≤ 0.05) in the small follicles. Serum concentrations of sodium, chloride, calcium and phosphorous were higher (p ≤ 0.05), while that of potassium lower (p ≤ 0.05) than corresponding concentrations in the follicular fluid. The concentrations of leucine and arginine were higher (p ≤ 0.05) in follicular fluid when compared with serum concentrations, while the reverse was true for other amino acids. In conclusion, this study is indicative of either low or high concentrations of certain biochemical metabolites, hormones, electrolytes and amino acids in small and large follicles for the individual roles that they play in the growth and development of follicles in the one‐humped she‐camel.     

Intrafollicular Concentrations of Steroid Hormones and PGF2α in Relation to Follicular Development in the Mares during the Breeding Season

The concentrations of androstenedione, estradiol-17β, progesterone and PGF_2α contained in the follicular fluid produced by the follicles in collected ovaries of mares that have had estrous phase during the breeding season were measured and analyzed the relation between the growth stage of follicles and the hormone levels in the follicular fluid. An ultrasonographic diagnostic instrument was used to measure the diameter of the follicles in order to categorize the follicles into three groups the following: 8 small follicles (from 1.0 to less than1.5 cm), 8 medium follicles (from 1.5 to less than 3.0 cm), and 8 large follicles (from 3.0 to 5.0 cm), respectively. The analysis of the follicular fluid in ovaries of estrous mares showed that the concentrations of androstenedione were significantly higher in the medium or large follicles than in the small follicles and the concentrations of estradiol-17β were significantly higher in larger follicles than in the small or medium follicles (P<0.05). The concentrations of progesterone and PGF_2α, on the other hand, did not significantly vary regardless of follicluar size. In the follicles within the mare ovaries that have had estrous stage, the concentrations of the hormones related the ovulation, namely androstenedione and estradiol-17β, were higher with larger follicles.     

Immunolocalization of steroidogenic cells in small follicles of the chicken ovary: Anatomical arrangement and location of steroidogenic cells change during follicular development

Previous anatomical and histochemical studies suggested that interstitial cells were the only steroidogenic cells in the theca layer of small follicles of the chicken ovary. However, the precise cellular site of steroid production in the small follicles is not certain. Therefore, our goal was to identify steroidogenic cells in small follicles (< 10 mm in diameter) of the chicken ovary which have not entered the follicular hierarchy by localizing steroidogenic enzymes with immunocytochemistry. Polyclonal antisera used were anti-cholesterol side-chain-cleavage cytochrome P450 (P450scc), anti-17-hydroxylase cytochrome P450 (P450c17), and anti-aromatase cytochrome P450 (P450arom) for pregnenolone-, androgen-, and estrogen-producing cells, respectively. Ovaries were collected 2 hr after oviposition and embedded in Paraplast after fixation with 4% paraformaldehyde, 10% formaldehyde, or Bouin's solution. Tissues were sectioned (4–6 μm) and sections were mounted on poly-L-lysine coated slides. Sections were incubated overnight at room temperature with each specific antiserum raised in rabbits against cytochrome P450 steroidogenic enzymes or normal rabbit serum as a control and were immunostained with an avidin-biotin-peroxidase complex. Immunoreactivity for the P450 enzymes was absent in the granulosa layer but was present in the theca layer of the small follicles (< 10 mm in diameter). Interstitial cells in the single theca layer of cortical follicles embedded in the ovarian cortex (less than 1 mm in diameter) contained P450scc and P450c17. Cells which contained P450arom, identified as aromatase cells, surrounded the interstitial cells in the theca layer. In small white follicles (approximately 1 mm in diameter), large white follicles (approximately 2–4 mm in diameter), and small yellow follicles (approximately 5–10 mm in diameter) which protruded from the surface of the ovary, the theca layer is divided into the theca interna and the theca externa. P450scc and P450c17 were localized in interstitial cells in the theca interna and externa whereas P450arom was localized in aromatase cells of the theca externa. With follicular development, more interstitial cells staining for P450scc and P450c17 appeared in the theca interna than in the theca externa whereas aromatase cells staining for P450arom were localized only in the theca externa. The distance between interstitial cells and aromatase cells within the theca layer increased as the follicles matured, resulting in a change in the anatomical relationship of steroidogenic cells. Our results of immunolocalization of cytochrome P450 steroidogenic enzymes in developing small follicles suggest that: 1) granulosa cells in small follicles are steroidogenically inactive; 2) steroids are produced in two distinct cell populations in the theca layer of small follicles, namely interstitial cells and aromatase cells; and 3) the anatomical relationship and location of interstitial cells and aromatase cells in the theca layer change with follicular maturation (a two-cell model for steroidogenesis in small follicles during follicular development).     

Ultrasonic imaging of equine ovarian follicles and corpora lutea

One of the most profound theriogenology applications of transrectal diagnostic ultrasonography in mares involves the imaging of ovarian follicles and corpora lutea. The resolving capabilities (frequency) and quality of the scanner directly affect the minimal size of a structure that can be imaged and the quality of the image. High-frequency scanners (5 or 7.5 MHz) of good quality can image a 2-mm follicle and the corpus luteum throughout its functional life. A low-frequency scanner (3 or 3.5 MHz) can image a 6-mm follicle and the corpus luteum for several days after ovulation. Equine follicles are excellent subjects for transrectal imaging because they are large, filled with fluid, and readily accessible. Event the small follicles (less than 10 mm) can be diagnostically important in evaluating whether ovarian infertility has occurred and whether the follicles are responding to treatment for follicular stimulation. The large, preovulatory follicles are of special interest. Averaged over a group of 79 periods, the following significant changes were found in the preovulatory follicle: increasing diameter, shape change from spherical to pear-shaped or conical, and increasing thickness of the follicular wall. No significant changes were found in the echogenicity (gray-scale value) of the wall or fluid. In retrospect, the diameter of the follicle seemed as useful for predicting impending ovulation as any of the other ultrasound criteria. The occurrence of ovulation is readily detected by the disappearance of a large follicle that was present at a recent previous examination. In addition, the ovulation site on the day of ovulation is detectable. In one study, the site was correctly identified in 24 of 24 mares. A small amount of residual follicular fluid can sometimes (7 of 10 in one study) be detected at the site of ovulation. The residual fluid usually disappears over a period of 0.5 to 20 hours. Subsequently, the developing corpus luteum may form a central nonechogenic area with peripheral luteinization or may remain uniformly luteinized. The central areas are of apparently vascular origin (blood or a component of blood) and become clotted and organized. In one study, approximately 50 per cent of the glands developed central areas exceeding 10 per cent of the size of the gland. The central areas began to develop on Day 0 or 1 and continued to enlarge until Day 2 or 3. The relative proportion of the gland containing a central clot decreases after Day 3, but the central area usually remains visible throughout diestrus.(ABSTRACT TRUNCATED AT 400 WORDS)     

驴,马卵泡发育及黄体形成的超声显像初步研究

本实验采用实时Ｂ型超声显像法，初步观察了１６头母驴和１７匹马发情期卵泡发育及黄体形成。卵泡为一轮廓完整的液性器官，卵泡液呈无回声（暗区）。卵泡壁在排卵前变薄，回声增强，轮廓更加清晰。７５％的马卵泡在排卵前２天由椭圆形变为梨形或锥形，大部分驴的卵泡始终保持椭圆形。９０％以上的驴和／或马的卵巢上有多个卵泡发育，多卵泡发育比单卵泡发育排卵迟缓。驴的最大卵泡从排卵前５天开始迅速增大，于排卵前１天达到最大（     

Expression of Vascular Endothelial Growth Factor Receptors in Bovine Cystic Follicles

Cystic follicles have excess fluid derived from blood flow in the theca interna of the follicle; therefore, the vasculature network is related to cystic follicle formation. Vascular endothelial growth factor (VEGF) is a potent stimulator of blood vessel permeability and angiogenesis. The aim of this study was to examine the expression of VEGF receptors proteins and mRNA in cystic follicles to elucidate the VEGF system in cystic follicles. The expression of protein for VEGF receptors; fms‐like‐tyrosine kinase‐1 (Flt‐1) and foetal liver kinase‐1 (Flk‐1) was detected by the immunohistochemical method. The mRNA expression of Flt‐1 and Flk‐1 in cystic follicles was determined by RT‐PCR. Concentration of oestradiol‐17β and progesterone in the follicular fluid of cystic follicles was determined using ELISA. Flt‐1‐ and Flk‐1 proteins were localized in granulosa and theca interna cells and endothelial cells of theca layers. The intensity of Flt‐1 and Flk‐1 immunoreaction was similar among cystic follicles with various ratios of oestradiol‐17β/progesterone concentrations. The expression of Flt‐1 and Flk‐1 mRNA was similar, regardless of the ratio of oestradiol‐17β to progesterone in follicular fluid. These results demonstrate that cystic follicles have both VEGF receptors in the granulosa and theca interna layers, which may be responsible for the increased permeability of microvessels, causing the accumulation of follicular fluid in cystic follicles.     

Ultrasonographic characteristics of the preovulatory follicle preceding and during ovulation in mares

Changes in appearance of preovulatory follicles were observed with real-time ultrasonography prior to and during ovulation in mares. Preovulatory follicles of 15 mares were scanned at < 1 hr intervals for 12 hr or more frequently if displaying signs of impending ovulation. If ovulation was not imminent at the end of 12 hr (n = 2), mares were removed from the trial. Mean follicular diameter decreased 13% from 30 minutes prior to ovulation until the beginning of ovulation. Fifteen to 77 minutes (mean = 41 min) prior to ovulation, a break in or a protrusion of the follicular wall toward the ovulation fossa was visualized in all follicles and was a consistent indicator of impending ovulation. A rapid decrease in size of follicles (ovulation) occurred within a period of 5 to 90 seconds (mean = 42 sec). Little or no fluid remained in the antrum following ovulation. An increase in echogenicity (whiteness) of the follicular wall and echogenic “spots” within the follicle were frequently visualized (13/13, 100% and 7/13, 54% respectively) prior to ovulation; however, prediction of time of ovulation could not be based solely on these individual changes.     

Follicular growth and estradiol concentrations in foaling and nonparturient mares

Plasma estradiol concentration and follicular development were evaluated daily during the first postpartum estrus and the subsequent cycle of five foaling mares. For comparison, one estrous cycle was monitored in the same fashion for five nonparturient mares. The first postpartum estrous cycles were shorter but similar to non-pregnant cycles in ovarian steroid production and follicular activity. However, estradiol production from postpartum follicles was lower per mm follicular diameter than from follicles in nonpregnant cycles (p<0.05).     

Insulin-like growth factor binding proteins in granulosa and thecal cells from bovine ovarian follicles at different stages of development

Because IGFBP inhibit IGF-stimulated cellular proliferation and differentiation, it is hypothesized that variations among IGFBP in individual follicles might contribute to the regulation of recruitment, selection, dominance, and turnover of ovarian follicles. Sources of IGFBP in fluid of bovine follicles are not well established; thus, objectives of this study were to determine levels of IGFBP binding activities and messenger RNA (mRNA) in granulosa and theca interna cells at different stages of follicular development (small [< 6 mm], medium [6 to < 8 mm], and large [> or = 8 mm]) and to characterize associations of these levels measured in the cells with levels of IGFBP and steroids in follicular fluid. Thecal and granulosa cells from large healthy follicles contained two- to twentyfold less (P < 0.05) IGFBP-2, -3, and -5 than cells from small, medium, and large atretic follicles. Thecal cells from small, medium, and large atretic follicles contained more (P < 0.05) IGFBP-3 and -4 than granulosa cells from these follicles, whereas granulosa cells from these follicles contained more IGFBP-2 activity than thecal cells. Differences in IGF binding activity were paralleled by differences in levels of mRNA for the respective IGFBP. Developmental differences in IGFBP activity in follicular fluid were positively associated with activity in granulosa and/or thecal cells, with the exception of IGFBP-4, which was low in fluid from large healthy follicles but markedly increased (mRNA and binding activity) in granulosa cells from these follicles. It is concluded that developmental changes in follicular fluid IGFBP-2 and -5 binding activities seem to be controlled in part by alterations in synthesis of these IGFBP by granulosa and thecal cells, whereas diminished IGFBP-4 in fluid from large healthy follicles occurs concomitantly with increased levels of IGFBP-4 mRNA and activity in granulosa cells, implicating posttranslational regulation by specific proteases.     

Characteristics of developing prolonged dominant follicles in cattle

In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.