Oxytocin and estradiol concentrations in follicular fluid as a means for the classification of large bovine follicles

Large antral follicles (13 to 20 mm in diameter) were collected from ovaries of 109 cows and 17 heifers that also had a regressed corpus luteum at slaughter. Thirty percent of the animals had been injected once with prostaglandin F(2)alpha 48 hours before slaughter. Follicles were divided into 3 groups based on estradiol and oxytocin concentrations in the follicular fluid: Group I follicles, estradiol>/=100 ng/ml and oxytocin<65 pg/ml (preovulatory and assumed pre-gonadotropin surge); Group II follicles, estradiol<100 ng/ml and oxytocin>/=65 pg/ml (preovulatory and assumed post-gonadotropin surge); and Group III follicles, estradiol<100 ng/ml and oxytocin<65 pg/ml (atretic follicles). Treatment with prostaglandin F(2)alpha significantly increased the number of viable granulosa cells and estradiol content in Group I follicles. The estradiol: progesterone ratio was significantly higher in Group I vs Groups II and III, but it was similar for Group II healthy follicles and Group III atretic follicles. To ascertain the classification of follicles, PGF(2)alpha was administered on Day 6 of the cycle to induce corpus luteum regression, and a GnRH analog was administered 24 hours later. At 23 hours after GnRH analog treatment, follicular oxytocin levels significantly rose to 103 pg/ml. Concomitantly, estradiol concentrations fell to below 100 ng/ml. This response was not evident by 13 h after injection of the GnRH analog. The results indicate that follicular estradiol and oxytocin concentrations may be used as a means for the physiological classification of large bovine follicles.     

Androgen metabolites in bovine follicular fluid

The values of C21-steroids, Delta4-androgens, estrogens as well as 5alpha-reduced steroids have been determined in follicular fluid obtained from superovulated and untreated cows. In the three cows treated with a hormone regimen to induce superovulation, the levels of progesterone and estradiol determined in 3 to 6 follicles per cow ranged from 65 to 448 ng/ml and 1.9 to 8.6 ng/ml, respectively while the concentrations of androstenedione and testosterone varied between 1.5 to 2.5 ng/ml. Low levels of dihydrotestosterone and androstane-3alpha, 17beta-diol (approximately 30 to 50% of Delta4-androgens) were found in the bovine follicular fluid. In untreated cows, the follicular steroid concentrations were divided into two groups on the basis of the ratio between estrogen and Delta4-androgen concentrations. In estrogen-rich follicles, the ratio of estrogens Delta4- androgens was higher than 1 and in estrogen-poor follicle, the ratio of estrogens Delta4- androgens was lower than 1. Pregnenolone, dehydroepiandrosterone, androst-5-ene-3beta, 17beta-diol, progesterone, androstenedione and testosterone levels were not significantly different in the two groups while the levels of estradiol and estrone were approximately 100-fold higher in the estrogen-rich group. The concentrations of 5alpha-reduced steroids particularly, dihydrotestosterone, androstane-3alpha, 17beta-diol and androsterone as well as their glucuronides which were found at values extremely low (under 1 ng/ml) were not significantly different in both groups. The results indicate that low levels of 5alpha-reduced steroids and their glucuronides are present in bovine follicular fluid and their concentrations remained fairly stable either in estrogen-rich or estrogen-poor groups.     

Immunosuppressive properties of follicular fluid from preovulatory horse follicles

Fluid was aspirated from the preovulatory follicles of mares before and 12, 24 and 36 h after intravenous administration of hCG. Follicular fluid significantly (P less than 0.001) reduced lymphocyte blastogenesis in vitro and, at a dilution of 1:100, fluid collected at 36 h after administration of hCG was significantly more suppressive (P less than 0.01) than fluid collected before 36 h. Suppression of blastogenesis was reduced by extracting the follicular fluid with ether or by charcoal treatment (P less than 0.01) or by heating at 56 degrees C for 30 min (P less than 0.05). Preincubation of lymphocytes with 2 of 5 follicular fluid samples expressed subsequent blastogenesis. Follicular fluid inhibited blastogenesis of T-cell growth factor (TCGF)-dependent Con A lymphoblasts (P less than 0.05) and the degree of inhibition was related to time of addition of the TCGF and time of collection of the follicular fluid. These results indicate that preovulatory follicular fluid in the mare is increasingly suppressive to lymphocytes as time of ovulation approaches and that this immunosuppression is associated with an alteration of the response to lymphokine stimulation.     

Isolation of inhibin from bovine follicular fluid

Bovine follicular fluid was used as a source for the isolation of gonadal inhibin, the activity of which was monitored by the dose dependent suppression of the FSH content of cultured pituitary cells. The procedures presented result in over 3000-fold purification of the starting material and the purified inhibin has an apparent molecular weight of 56000. The purified inhibin can be dissociated under reducing conditions into two subunits with molecular weights of 44000 and 14000 daltons.     

Effects of bovine follicular fluid on maturation of bovine oocytes

Three experiments were conducted to determine the effects of follicular fluid and media on bovine oocyte maturation. Experiments 1 and 3 test the effects of follicular fluid obtained at different times after the LH surge on bovine oocyte maturation in vitro, while Experiment 2 was designed to compare TALP and Medium 199 as serum-free maturation media. Bovine follicular fluid (BFF) was obtained from preovulatory follicles either before (0 h BFF) or at 4, 8, 12 or 20 h after a GnRH-induced LH surge. Oocytes were obtained from follicles 1 to 6 mm in diameter from ovaries retrieved from a slaughterhouse. In Experiment 1, both 0 h and 4 h BFF inhibited resumption of meiosis, whereas BFF collected at 8, 12 and 20 h did not. When oocytes were cultured in media that contained equal portions of 0 and 8 h BFF, meiosis was not inhibited. In Experiment 2, Medium 199 supplemented with bovine serum albumin (BSA) was superior to Tyrode's medium with albumin, lactate and pyruvate for oocyte maturation. In Experiment 3, a higher percentage (P<0.05) of oocytes cultured for 18 h in 40% 20 h BFF in Medium 199 reached Metaphase-II (64%) than those cultured in 0 h BFF (41%) or control medium (39%). There was a transient meiotic arrest due to 0 h BFF as evidenced by the higher percentage of oocytes with germinal vesicles at 8 h of incubation (35% with 0 h vs 20% with 20 h; P<0.05). Furthermore, expansion of cumulus cells was induced in 8 and 20 h BFF, but not 0 h BFF.     

Steroid hormone concentrations in the fluid of bovine follicles relative to size, quality and stage of the oestrus cycle

Eight hundred and seven bovine antral follicles from 2 mm to 20 mm in diameter were dissected free of stromal tissue, measured, qualified and divided into 36 groups according to size, quality and stage of cycle. The follicular fluid was collected and assayed by RIA for oestradiol-17beta, testosterone and progesterone. The steroid hormone concentrations vary with follicular size, degree of atresia and stage of the cylce. Non-atretic follicles of less than 8 mm are generally androgen-dominated and non-atretic follicles of more than 11 mm are oestrogen-dominated. Follicles betwen 8 mm and 11 mm are intermediate in this respect. Degeneration leads to a gradual decrease of oestradiol-17beta and testosterone concentration and increase of progesterone. It is suggested that the ratio of oestradiol-17beta/testosterone and oestradiol- 17beta/progesterone and oestradiol-17beta/testosterone + progesterone cannot generally be used to discriminate between non-atretic and atretic follicles. Large follicles present during the early luteal stage contain as much oestradiol-17beta in the follicular fluid as large follicles during the follicular stage, whereas large follicles of the luteal stage contain only 15% of the maximal amount of the latter's. This and other presented data support the statement that follicles present during the early luteal, late luteal and follicular stages of the cycle belong to different groups of growing follicles. It has been concluded that groups of macroscopically qualified follicles can be distinguished from each other by the steroid hormone concentration in the follicular fluid. It is therefore possible to predict the hormonal environment of the oocyte in any individual follicle of a defined size and quality.     

Relationship between concentrations of immunoreactive insulin-like growth factor-I in follicular fluid and various biochemical markers of differentiation in bovine antral follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in follicular estradiol and gonadotropin receptors during development of bovine antral follicles

It was hypothesized that growth divergence of dominant and subordinate follicles during Wave 1 and growth termination of the dominant follicle would be associated with changes in the number of gonadotropin receptors on granulosa cells and estradiol in follicular fluid. To test this hypothesis, follicular development of 16 Holstein heifers was monitored by ultrasound, and follicles were collected on Days 2,4,6 and 10 (Day 0 = ovulation). Dominant follicles were compared across days, whereas dominant and largest subordinate follicles were compared on Days 2 and 4 only. The numbers of LH and FSH receptors on the granulosa cells of dominant follicles did not differ significantly over Days 2, 4, 6 and 10. In contrast, concentrations of estradiol in follicular fluid decreased (P < 0.05) from Days 2 to 10 (373 +/- 150 to 42 +/- 12 ng/ml) and concentrations of progesterone in follicular fluid increased (P < 0.05) from Days 2 to 10 (12.2 +/- 2.3 to 24.4 +/- 4.8 ng/ml). Correspondingly, the ratio of estradiol:progesterone in the dominant follicles decreased (P < 0.05) from Days 2 to 10. Comparisons between dominant and subordinate follicles indicated greater (P < 0.05) estradiol concentrations in the dominant follicle on Day 2, but the number of gonadotropin receptors was not different until Day 4. Thus, differences in concentrations of follicular fluid estradiol, but not numbers of granulosa cell gonadotropin receptors, were associated with the early growth divergence of dominant and subordinate follicles (Day 2) and the eventual growth termination of the dominant follicle (Day 10). Late divergence (Day 4) was associated with higher gonadotropin receptor numbers and follicular estradiol concentrations in the dominant than in the subordinate follicles. These results indicate that an increase in estradiol productivity of the selected dominant follicle occurred before an increase in the number of gonadotropin receptors.     

The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes

The ovary of cattle contains thousands of oocytes which are enclosed primarily in the preantral follicles. Methods of culturing preantral follicles are now being developed. The aim of this study was to investigate the effect of the size of bovine preantral and early antral follicles and culture media on their in vitro growth. Individual follicles isolated by microdissection of the ovarian slices were sorted into the following size classes: 75 to 124, 125 to 174, 175 to 224, 225 to 274, 275 to 324 and > or = 325 microns. The follicles were cultured individually in TCM 199 + fetal calf serum (FCS) + supplements (FSH, estradiol-17 beta, insulin, transferrin, sodium selenite, sodium pyruvate, 1-glutamine and hypoxanthine) or in Menezo B2 + FCS + supplements (Experiment 1) and in TCM 199 + steer serum (SS) with or without additional supplements (Experiment 2). The total number of isolated follicles of different size classes was similar in heifers and cows. No significant difference in the growth rate of follicles of different sizes was seen in the 2 media (TC 199 and B2). However, the culture of follicles in the TCM 199 that was supplemented only with SS and contained no other additives significantly reduced follicular survival and growth in comparison with follicles cultured in the supplemented medium. The survival time of follicles was related to their initial size at the beginning of culture. The longest period of growth was for follicles 275 to 324 microns in diameter (i.e., 10.7 +/- 5.7; 12.1 +/- 6.2 and 12.2 +/- 2.7 d, respectively, for culture in supplemented Menezo B2, TCM 199 + FCS and TCM 199 + SS). Survival and growth of some follicles was maintained for 23 d.     

Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovine spermatozoa

Bovine epididymal and ejaculated spermatozoa were incubated for 22 and 9.5 h respectively, in a chemically defined medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. Addition of bovine follicular proteoglycan or chondroitin sulfates ABC significantly increased the incidence of acrosome reaction. The stimulatory effects of the proteoglycan or chondroitin sulfates were negated by exposure to the enzyme chondroitinase ABC. Viability of spermatozoa was not affected by the various experimental treatments. Transmission electron microscopy of spermatozoa showed that vesiculation had occurred between the plasma and outer acrosomal membrane. These results suggest that proteoglycan present in follicular fluid at the time of ovulation may promote the acrosome reaction which precedes the ability of sperm to fertilize an ovum.     

Relationship of macroscopic appearance of the surface of bovine ovarian follicles concentrations of steroids in follicular fluid, and maturation of oocytes in vitro

The relationship among opaqueness of the surface of bovine ovarian follicles, concentrations of follicular steroids, and capacity of oocytes to achieve nuclear maturation in vitro was examined in this study. Follicles greater than or equal to 5 mm in diameter were classified as clear (n=68) or opaque (n=72) based on their surface appearance. An oocyte and follicular fluid (FF) were removed from each follicle. Each oocyte was cultured, and the concentration of estradiol (E), progesterone (P), and testosterone (T) was determined for each sample of FF. Oocytes that extruded the first polar body by 30 h in culture were considered mature. All other oocytes were immature. More (p less than 0.05) mature oocytes came from clear (56%) than opaque follicles (29%). Clear follicles had lower concentrations of E (p less than 0.05) and P (p less than 0.10) in FF than opaque follicles. Follicles with mature oocytes had greater (p less than 0.05) concentrations of P than follicles with immature oocytes. Follicles were separated into three categories based on ratio of P:E in FF: high = P:E greater than or equal to 10, medium = P:E greater than or equal to 1 less than 10, and low = P:E less than 1. The percentage of mature oocytes from clear follicles was similar for high (64%), medium (48%), and low (57%) P:E groups; however, the percentage of mature oocytes from opaque follicles was greater (p less than 0.05) for the high (59%) than for the medium (21%) or low (19%) P:E groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of follicle size and quality on the ability of follicular fluid to support cytoplasmic maturation of bovine oocytes

Recent studies have shown that the developmental capacity of in vitro-matured oocytes is affected by the origin of follicular fluid (FF) supplemented to the maturation medium. The aims of this study were (1) to determine if follicle size and quality would influence the capacity of FF to support bovine oocyte maturation and (2) to determine if fetal calf serum (FCS) and FF had an additive effect when added together to the maturation medium. Follicular fluid collected from 108 follicles was classified according to size (<6, 6–8, >8 mm in diameter) and quality (healthy, early atretic, and atretic). Quality, first determined by mitosis/pycnosis ratios in granulosa cell smears, was subsequently confirmed by insulin-like growth factor binding protein (IGFBP) patterns and estradiol concentrations. While most small- or medium-sized follicles showed some atresia (88% and 67%, respectively), fewer of the large follicles were atretic (30%). In experiment 1 bovine oocytes (n = 2,152) were matured either in TCM199 alone, with 10% FCS, or with 10% FF from the following follicle types: small healthy (SH); small early atretic (SEA); small atretic (SA); medium healthy (MH); medium early atretic (MEA); medium atretic (MA); large healthy (LH); large early atretic (LEA); and large atretic (LA). Following IVM, oocytes were fertilized and subsequently cultured in synthetic oviduct fluid (SOF). Day 8 blastocyst yields were 23% in TCM199 alone; 37% in TCM199 plus FCS; and, in medium supplemented with FF, SH, 36%; MH, 32%; LH, 30%; SEA, 21%; MEA, 26%; LEA, 28%; SA, 32%; MA, 33%; and LA, 38%. All FF from healthy or atretic follicles resulted in significantly improved blastocyst yields compared to M199 alone (P < 0.05) However, FF from early atretic follicles irrespective of size did not yield a significant improvement. In experiment 2 we examined the effect of addition of FF-LH and serum together to the maturation medium. In terms of blastocyst yield, no additional benefit was observed when TCM199 was supplemented with 10% FCS + 10% FF (33%) compared to 10% FCS or FF alone (35% and 30%, respectively). The efficacy of FF as a supplement to the maturation medium to improve cytoplasmic maturation appears to vary with follicle quality but not size. However, in general, the addition of 10% FF or FCS to the maturation media resulted in a similar blastocyst yield with no additional improvement when media was supplemented with both FCS and FF. © 1996 Wiley-Liss, Inc.     

Chemotactic response of frozen-thawed bovine spermatozoa towards follicular fluid

The aim of this study was to verify whether cattle spermatozoa respond by chemotaxis to follicular fluid (FF). The experimental conditions were defined to maintain a frozen-thawed sperm population with great motility and capacitation, and lesser sperm agglutination. Several sperm preparation conditions were studied: sperm separation from the seminal plasma by Sephadex column or migration-sedimentation, incubation under capacitating conditions in the presence or absence of a superficial layer of mineral oil, and different pH of the culture medium. The percentage of motile and agglutinated spermatozoa was determined in plate dishes under inverted phase contrast microscope. The percentage of capacitated spermatozoa was calculated as the difference between the percentages of acrosome reacted spermatozoa with and without lysophosphatidylcholine stimulation. The most ideal experimental conditions to evaluate chemotaxis in frozen-thawed cattle spermatozoa were: to separate the cells from the seminal plasma by migration-sedimentation and to incubate them under oil, in culture medium at pH 7.2, for less than 2h. The chemotaxis assays were conducted with spermatozoa treated as mentioned above which were confronted to several dilutions of FF (1:10(3), 1:10(4), 1:10(5), 1:10(6)) in a chemotaxis chamber by videomicroscopy and computer image analysis. A subpopulation of capacitated spermatozoa ( approximately 10%) that responded chemotactically to a concentration gradient generated by FF (1:10(4) to 1:10(5)) was observed. Since cryopreserved spermatozoa are regularly used to artificially inseminate the cows, the sperm chemotactic response towards FF would be potentially used to diagnose the bull sperm sample or to select the spermatozoa in the most functional state.     

Localization and detection of ovarian follicular fluid protein in follicles of human ovaries.

Human ovarian follicular fluid protein has been partially purified and the active fraction designated as hGF2. Using specific polyclonal antiserum to hGF2, it was observed to be localized immunohistochemically in the granulosa cells of medium but not large follicles of human ovary. The hGF2 levels were estimated by ELISA in serum and follicular fluid of 10 gonadotropin-stimulated women recruited for IVF-ET programme. The results revealed a 3-fold increase in the concentration of hGF2 in follicular fluid compared to that in serum of these patients. These data indicate that the protein is secreted by granulosa cells and plays an important role in the regulation of follicular maturation and ovulation.     

Relationships between histological signs of atresia, steroids in follicular fluid, and gonadotropin binding in individual bovine antral follicles during postpartum anovulation

Two experiments were conducted to determine the relationship between histological signs of atresia, gonadotropin binding, and steroids in fluid of medium-sized bovine follicles during postpartum anestrus. In Experiment I, ovaries of 21 cows were removed on Days 7, 14, 28, 42, or 56 after parturition. In Experiment II, ovaries of 29 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH; 500 ng/5 ml saline) injections given every 2 h via jugular cannulas. Two to 10 follicles, 4.0-7.9 mm in diameter, were removed per pair of ovaries. Follicles were classified as normal, intermediate, atretic, or luteinized-atretic, depending on their micromorphology. In both Experiments I and II, follicles classified as normal had 50-80% lower (p less than 0.05) concentrations of progesterone and 2- to 7-fold greater (p less than 0.05) concentrations of estradiol than atretic follicles. However, concentrations of androstenedione and gonadotropin-binding sites were similar in normal and atretic follicles. Atretic follicles had degenerative granulosa with several pyknotic nuclei, thick theca, and little distinction between theca and granulosa. Intermediate follicles showed slight signs of degeneration and had 2- to 3-fold greater (p less than 0.05) concentrations of progesterone than normal follicles. Concentrations of estradiol did not differ (p greater than 0.10) between normal and intermediate follicles. Equal proportions of normal and atretic medium-sized follicles were located on the ovaries bearing the corpus albicans from pregnancy (CAP).(ABSTRACT TRUNCATED AT 250 WORDS)