Superovulation and embryo recovery from peripubertal Holstein heifers.

The use of peripubertal donors in embryo transfer (ET) programs presents significant opportunity to accelerate genetic gain in domestic livestock by reducing the generation interval. These studies were designed to evaluate feasibility of superovulation and embryo recovery in peripubertal heifers (starting at 7.8 months of age), and to determine whether subsequent reproductive and lactational performance of donor heifers were impaired. Study 1 utilized 10 pairs of contemporary full-sibling heifers in which one heifer in each pair was assigned to receive a superovulation regimen and her full-sibling contemporary received placebo. Treated heifers were artificially inseminated at estrus and embryos were flushed transcervically 4-6 days later. Based on recovery of oocytes and/or embryos, 9 of 10 heifers responded to the hormonal regimen and 12 total embryos were recovered. Seven embryos (58%) were transferred into recipients resulting in five pregnancies. Control and treated heifers remained in the herd and were bred at a natural estrus by AI at 15 months of age. Lactation records, i.e., 305 days mature equivalent (305 d ME) were obtained, and all animals were evaluated for udder conformation traits between 32 and 38 months of age. Reproductive traits (age at first calving and days to conception) and lactational traits of heifers subjected to embryo transfer and their non-treated full-siblings did not differ (P > 0.05). Study 2 was conducted to establish the commercial feasibility of hormonally programming peripubertal heifers ranging in age from 7.8 to 9.9; 10 to 11.9; 12 to 13.9 and >/= 14 months. In total, 3982 embryos were recovered from 520 heifers, with 2419 (60.7%) of those categorized as viable (transferable). The number of ova/embryos obtained per flush (5.6 +/- 1.0) and the number of transferable embryos (2.8 +/- 0.5) was reduced (P < 0.05) in heifers of age 7.8-9.9 months compared to all other age groups. There was no difference (P > 0.05) in the number of ova/embryos recovered (7.8 +/- 0.3), or the number of transferable embryos (4.8 +/- 0.2), among heifers that were >/=10 months of age. The number of unfertilized ova did not differ by age, however, more degenerate embryos tended to be recovered from heifers <10 months of age compared to heifers >/=14 months of age. These data indicate that transferable embryos can be safely recovered from heifers beginning at 10 months of age without compromising subsequent reproductive or lactational performance of the donor.

Identification of a heparin-binding protein in bovine seminal fluid as tissue inhibitor of metalloproteinases-2.

Presence or absence of three distinct bovine seminal heparin-binding proteins (21-31 kDa) recognized in sperm extracts by a monoclonal antibody, M1, is a diagnostic indicator of fertility differences among bulls producing normal semen. We recently identified a 31 kDa fertility-associated antigenin bovine seminal fluid as a unique DNase I-like protein. We now report purification and identification of a 24 kDa seminal heparin-binding protein (HBP-24) recognized by M1. N-terminal microsequence analysis of HBP-24 purified from seminal fluid yielded 20 amino acid residues that displayed 90% identity to the N-terminus of a bovine metalloproteinase inhibitor identified as tissue inhibitor of metalloproteinases-2 (TIMP-2). A single immunoreactive band migrating at 24 kDa was detected in Western blots of cauda epididymal sperm extracts following incubation with purified seminal heparin-binding proteins and subsequent washing in vitro, indicating TIMP-2 bound to sperm membranes. Expression of TIMP-2 mRNA was detected by RT-PCR in bovine bulbourethral gland, prostate, and seminal vesicles. Mobility of the 24 kDa heparin-binding protein increased under nonreducing SDS-PAGE to approximately 21 kDa, characteristic of the reported molecular mass of TIMP-2. To our knowledge, this is the first report of TIMP-2 binding to spermatozoa and of TIMP-2 mRNA expression in bovine accessory sex glands. These results corroborate previous reports regarding the site of production of heparin-binding proteins that are related to bull fertility, and suggest that TIMP-2 influences fertility of bulls, either through inhibition of metalloprotease activity in semen or via undefined activities independent of matrix metalloproteinase (MMP) inhibition.

Artificial insemination outcomes in beef females using bovine sperm with a detectable fertility-associated antigen.

In this study, semen samples from 25 bulls that had passed a breeding soundness evaluation were analyzed for the presence or absence of a 31-kDa protein, known as fertility-associated antigen (FAA), on spermatozoal membranes. Eighteen bulls had FAA on sperm (FAA-positive) and seven were devoid of FAA on sperm (FAA-negative). A single ejaculate from each bull was extended and frozen with 25 to 30 x 10(6) sperm in .5-mL straws. Crossbred replacement heifers (n = 865) were estrus-synchronized and artificially inseminated either at timed AI or 12 h after they were detected in estrus. Mature cows (n = 285) were inseminated 12 h after they were detected in estrus during a 45-d AI period. Pregnancy rates (pooled) to first AI service for females (n = 764) inseminated with FAA-positive sperm were 65.6% and were 49.7% for females (n = 386) inseminated with FAA-negative sperm (P < .005). Among the estrus-synchronized replacement heifers, pregnancy rates to synchronized AI service for heifers (n = 550) inseminated with FAA-positive sperm were 62% and were 45.7% for heifers (n = 315) inseminated with FAA-negative sperm (P < .005). These data indicate that pregnancy rates to first AI service at spontaneous and synchronized estrus are higher when using semen from bulls with detectable FAA on spermatozoal membranes compared to semen from bulls devoid of FAA on membranes. Fertility-associated antigen is an important determinant for fertility potential of sperm from bulls to be used in AI breeding programs.

Purification and characterization of fertility-associated antigen (FAA) in bovine seminal fluid.

Heparin-binding proteins (HBP) recognized by a monoclonal antibody (M1) are produced by male accessory sex glands and bind to distinct regions of ejaculated bull sperm. Immunoblots of sperm proteins probed with M1 identified HBP variants of approximately 31-, 24-, and 21.5-kDa that were associated with increased fertility of bulls. The purpose of this study was to identify the 31-kDa HBP known as fertility-associated antigen (FAA). FAA was isolated by heparin-affinity chromatography and reversed-phase high performance liquid chromatography near homogeneity. Biochemical characterization indicated that FAA was an unglycosylated, basic protein. FAA protein was detected in seminal vesicle and prostate gland homogenates, and FAA extracted from sperm membranes by treatment with hypertonic media was identical biochemically to seminal fluid-derived FAA. N-terminal sequence analysis of purified FAA yielded a 26 amino acid sequence (L K I X S F N V R S F G E S K K A G F N A M R V I V) with 73% identity to a recently identified human deoxyribonuclease (DNase) I-like protein. Two internal amino acid sequences generated from lys-C digested FAA were 85% and 92% identical to the same DNase I-like protein. In conclusion, we have identified a bovine seminal heparin-binding protein that binds to sperm and is indicative of bull fertility as being similar to the family of DNase I-like proteins. These data demonstrate the presence of a novel DNase I-like protein in bull accessory sex glands and form the groundwork for the identification of a candidate genetic marker for fertility of bulls.

Fertility-associated antigen on bull sperm indicates fertility potential.

A 30-kDa heparin-binding protein named fertility-associated antigen (FAA) was identified in sperm membranes of beef bulls with greater fertility potential. In a survey of 2,191 beef bulls, 88% had FAA present in sperm membranes (FAA-positive), and 12% were FAA-negative. In the first study, 54 Santa Gertrudis and 51 Santa Cruz bulls were grouped (1 to 14 bulls per group) according to FAA profiles and were bred to 2,403 cows at ratios of 1 bull: 25 cows. Fertility for 14 groups of FAA-positive bulls averaged 88%, whereas three groups of FAA-negative bulls impregnated 79% of the cows. Thus, FAA-positive bulls were nine percentage points more (P < .01) fertile than FAA-negative bulls. In the second study, 2-yr-old Santa Cruz bulls (n = 26) were grouped according to FAA profiles and serving capacity. The fertility of the group of 12 high-serving-capacity, FAA-positive bulls was 87% of 270 cows. The group of six FAA-negative bulls with high serving capacity impregnated 78% of 143 cows. Among the groups of bulls with high serving capacity, FAA-positive bulls were nine percentage points more (P < .05) fertile than FAA-negative bulls. The group of eight FAA-positive bulls with low serving capacity impregnated the least (P < .01) percentage (69%) of 238 cows. Serving capacity of bulls should be considered when optimizing fertility potential. Among bulls with acceptable physical characteristics and serving capacity, determination of FAA profiles in sperm can be used as a tool to identify subfertile bulls.

Localization of a heparin-binding protein to distinct regions of bovine sperm.

Heparin-binding proteins (HBP) in bull seminal fluid bind to epididymal sperm membranes at ejaculation. Peptides recognized by a monoclonal antibody (M1) correspond to proteins identified in complexes that have the greatest affinity for heparin and are present on sperm from bulls with higher fertility. Presence of specific HBP on sperm regulates the ability of sperm to bind heparin, and heparin binding to sperm correlates with the fertility potential of a bull. In these studies, the interaction of HBP with sperm from 10 bulls of proven fertility was analyzed by immunofluorescence of M1 to determine the localization of heparin-binding proteins during capacitation, and the fluorescent binding patterns were compared to bull nonreturn rates. Immunofluorescent localization of M1 binding sites revealed the existence of specific membrane domains containing HBP in acrosomal and postacrosomal regions of ejaculated but not in epididymal sperm. Monoclonal antibody recognition of HBP localized on membranes of sperm revealed variable binding patterns of M1 to the acrosomal region in sperm from bulls of known fertility. Regression analysis indicated a negative relationship between sperm displaying exclusively acrosomal fluorescence and bull nonreturn rate. These data indicate that HBP bind to sperm in distinct patterns, one of which differed among bulls of varying fertility, and indicate no apparent relocalization of these sites during cellular changes that occur in preparation for fertilization.

Monoclonal antibody detection of heparin-binding proteins on sperm corresponds to increased fertility of bulls.

A monoclonal antibody (M1) was produced against seminal fluid heparin-binding proteins (HBP) from a vasectomized bull. In the first part of this study, the presence of HBP in sperm or seminal fluid was determined for 53 bulls with an ELISA using M1. Bulls (8 to 18 per pasture) were bred to 1,114 cows at ratios of 1 bull:25 cows. Bulls with detectable HBP on sperm membranes were 11 percentage points more fertile than bulls with undetectable HBP in sperm membranes. In the second part of this study, three sperm, membrane HBP approximately 30, 24, and 21.5 kDa were identified with Western blots using M1. Santa Gertrudis bulls (n = 64) were bred to 1,354 Santa Gertrudis cows in groups with 2 to 11 bulls. Bulls with those three HBP (Group A) or a single 30-kDa HBP (Group B) in sperm membranes had the greatest fertility, ranging from 74.4 to 89.9% (mean = 81.5%) of the palpated cows that were pregnant. Bulls with the 21.5- and 30-kDa HBP (i.e., the 24-kDa HBP was absent; Group C) had a reduced fertility of 61.3%. Bulls without detectable HBP (Group D) resulted in 41.9% of 186 cows palpated pregnant. Bulls in Groups A and B were more (P < .01) fertile than all other groups. In conclusion, the presence of HBP in sperm membranes was indicative of the fertility potential of bulls.

Fertility of range beef bulls grouped according to presence or absence of heparin-binding proteins in sperm membranes and seminal fluid.

Trials were performed to determine the relationship of heparin-binding proteins (HBP) to fertility of bulls. Red Angus (142), Santa Gertrudis (59), Gelbvieh (59), and Santa Gertrudis x Gelbvieh (40) bulls were identified according to the presence or absence of the greatest affinity HBP (HBP-B5) on sperm membranes and in seminal fluid. Nine to 20 bulls with the same HBP-B5 profiles were assigned to pastures with Santa Gertrudis cows at a ratio of 1 bull:25 cows. Fertility for Group 1 (80 bulls with HBP-B5 in sperm membranes but undetectable HBP-B5 in seminal fluid, in six pastures) was 82% pregnant of 1,692 cows. Group 2 bulls (48 bulls with HBP-B5 detectable in seminal fluid and in sperm membranes, in four pastures) impregnated 67% of 919 cows. Fertility for Group 3 (37 bulls with HBP-B5 in seminal fluid but undetectable HBP-B5 in the sperm membranes, in three pastures) and Group 4 (56 bulls with undetectable HBP-B5 in seminal fluid and sperm, in four pastures) was 63% pregnant of 747 and 1,208 cows, respectively. Group 1 had an average of 17% greater fertility compared with Groups 2, 3, and 4 (P < .05). In conclusion, groups with the greatest affinity HBP-B5 in sperm membranes but not in seminal fluid had greater fertility than did groups with other HBP-B5 profiles.

Binding of bovine follicular fluid glycosaminoglycans to fibronectin, laminin and low-density lipoproteins.

Interactions of bovine follicular fluid glycosaminoglycans (GAGs) with extracellular matrix (ECM) components fibronectin and laminin and with low-density lipoproteins (LDL) were examined using affinity chromatography. Glycosaminoglycans from small (diameter less than 5 mm) and large (diameter 11-20 mm) follicles were isolated from follicular fluid. The dermatan sulphate or heparan sulphate from small or large follicles was applied to Fn-, Lm- or LDL-Sepharose columns. Portions of each fraction of the bound or unbound GAG were then subjected to gel filtration h.p.l.c. for quantification. The binding interaction between dermatan sulphate and fibronectin was significantly greater than between heparan sulphate and fibronectin (P less than 0.05); the binding interaction between GAGs from small follicles and fibronectin was significantly greater than between GAGs from large follicles (P less than 0.05). The binding interaction between GAGs from small follicles and laminin was significantly greater than for GAGs from large follicles (P less than 0.05). Dermatan sulphate from small follicles bound to fibronectin (42%), laminin (36%) and LDL (14%) and that from large follicles bound to fibronectin (14%), laminin (23%) and LDL (14%). Heparan sulphate from small follicles bound to fibronectin (17%), laminin (15%) and that from large follicles bound to fibronectin (13%), laminin (10%) and LDL (6%). These results suggest that dermatan sulphate, but not heparan sulphate, from follicles at different stages of development exhibit a varied ability to interact with components of the ECM. Both substances bound to LDL comparably in small amounts.

Predicting fertility of dairy bulls by inducing acrosome reactions in sperm with chondroitin sulfates.

Bull spermatozoa were incubated with 0 or 50 micrograms/ml chondroitin sulfates to induce acrosome reactions in vitro. Data were computed as percent increase in acrosome reactions elicited by chondroitin sulfates compared with control samples for each replicate. Data from 8 bulls with known nonreturn rates served as basis to predict nonreturn rates of 18 bulls entering an AI lineup. The average assay CV was 16.1% for all 26 bulls. Correlation coefficients of polynomial regression lines computed between actual nonreturn rates and percent increases in acrosome reactions in response to chondroitin sulfates were .98 and .81 for the 8 reference bulls and combined data of all 26 bulls, respectively. It is concluded that a laboratory assay using chondroitin sulfates to induce acrosome reactions in sperm can be used to predict relative fertility of bulls.

Follicular fluid glycosaminoglycans inhibit degradation of low-density lipoproteins and progesterone production by porcine granulosa cells.

Porcine granulosa cells were incubated with commercially available glycosaminoglycans (GAGs) or GAGs purified from porcine follicular fluid to evaluate the effects of GAGs on degradation of low-density lipoproteins (LDL) and progesterone production. Commercially available heparin and chondroitin sulfates (CS) as well as follicular CS and heparan sulfate (HS) inhibited degradation of LDL in dose-dependent manners. Doses of follicular CS and HS required to inhibit 50% of the LDL degradation corresponded to concentrations found in follicular fluid (less than 1 mg/ml). Progesterone production was also inhibited in a dose-dependent fashion by follicular GAGs at concentrations found in follicles. The ability of the follicular GAGs to inhibit degradation of LDL could represent a mechanism by which the utilization of LDL-derived sterol is temporarily restricted following permeabilization of the ovulatory follicle. Follicular GAGs may also modulate utilization of apoprotein E-containing high-density lipoproteins in unruptured follicles.

Influence of stage of cycle, corpus luteum location, follicle size, and number of large follicles on estradiol-17 beta concentrations in bovine follicles.

Concentrations of estradiol-17 beta in follicular fluid were correlated to follicular size, stage of estrous cycle, location of corpus luteum, and presence of large follicles. Paired ovaries were obtained from 481 nonpregnant cows at slaughter and follicles were classified as ipsilateral or contralateral to the corpus luteum. Follicular fluid estradiol-17 beta concentrations from 2494 small, 1485 medium, and 396 large follicles were quantified by radioimmunoassay. Stage of estrous cycle was estimated by visual examination of the corpus luteum. Follicles in stage 1 of the estrous cycle (d 1 to 4) had the highest estradiol-17 beta concentration and the smallest mean follicular diameter. Location of follicles relative to the corpus luteum had no influence on estradiol-17 beta concentrations. As follicular size increased, concentration of estradiol-17 beta also increased. The presence of a single large follicle did not affect the concentration of estradiol-17 beta in medium or small follicles. In contrast, if multiple large follicles occurred in the same cow, concentrations of estradiol-17 beta were significantly lower in medium but not small follicles.

Comparisons of the ability of follicular fluid glycosaminoglycans and chemically desulfated heparin to compete for heparin-binding sites on granulosa cells.

Glycosaminoglycans (GAGs) were purified from bovine follicular fluid, and their effectiveness to compete for heparin-binding sites in granulosa cells was evaluated. The GAGs dermatan sulfate (DS) and heparan sulfate (HS) were purified by anion-exchange high-performance liquid chromatography. Approximately 5 micrograms of protein from suspensions of bovine granulosa cells were incubated with 101 pmoles of [3H]heparin and 0.01-5.0 mg/ml of HS or DS for 2 h at 37 degrees C in 40 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.35. Heparan sulfate obtained from small and medium follicles displaced [3H]heparin in a dose-dependent manner from 0.1 to 5 mg/ml, but HS from large follicles did not displace [3H]heparin. The DS obtained from small, medium, and large follicles displaced [3H]heparin in a dose-dependent manner, and the potency of the DS to displace [3H]heparin increased as the size of the follicles from which the DS was purified increased. Those results were independent of the maturational state of the granulosa cells. In a separate experiment, heparin (17.1% sulfate) was N-desulfated (11.8%), and the desulfated heparin did not displace [3H]heparin. It was concluded that the effectiveness of follicular HS and DS to compete for heparin-binding sites on granulosa cells was dependent on the maturation of the follicle from which the fluid was obtained rather than on the source of granulosa cells. The binding interaction of the GAGs relies, to some extent, on the presence and positions of sulfate moieties.

Purification of glycosaminoglycans from bovine follicular fluid.

The follicular glycosaminoglycans, dermatan sulfate and heparan sulfate, are not available in sufficient quantities for detailed analytical studies or to use as reagents in cell cultures. The purpose of this study was to purify follicular glycosaminoglycans simultaneously from bovine follicular fluid. The use of a chloroform:methanol extraction of 10 ml of fluid resulted in recoveries of at least 90% of the glycosaminoglycans, otherwise an insoluble product resulted. Heparan sulfate and dermatan sulfate eluted at .11 and .46 M NaCl, respectively, when subjected to HPLC with an anion exchange column. Fluid from small follicles allowed to "age" in the ovaries exhibited marked reductions in the concentrations of glycosaminoglycans. Aging of the fluid from large follicles resulted in greater glycosaminoglycan concentrations and appearance of glycosaminoglycans reflecting other charge distributions. Known amounts of purified follicular fluid heparan sulfate and dermatan sulfate were applied to a gel filtration HPLC column, and corresponding integrated peak areas were plotted in relation to the glycosaminoglycan concentrations. Slopes of the lines for commercial chondroitin sulfate and follicular dermatan sulfate were not significantly different, but the slope of the line for follicular heparan sulfate was 13.5-fold greater than the slope for commercial heparin.

Chemical characteristics of follicular glycosaminoglycans.

The two glycosaminoglycans found in FF, DS and HS were isolated from fluid of atretic and non-atretic bovine follicles and simultaneously separated by anion exchange high performance liquid chromatography. Heparan sulfate eluted with the apex of the peak at .071 M NaCl. Dermatan sulfate eluted with the apex of the peak at .353 M NaCl. The concentrations of DS and HS were greater in atretic follicles compared to non-atretic follicles and decreased as follicles size increased, as previously reported. The proportion of DS was approximately 90% of the total glycosaminoglycan concentration of all FF samples. The ratio of galactosamine to glucosamine in DS increased as follicle size increased, being 3.44 +/- .35, 7.65 +/- 2.4, and 15.9 +/- 3.8 (P less than .05) for small, medium and large follicles, respectively.

Reproductive performance of dairy cows with cystic ovaries following administration of Procystin.

Eighty-five postpartum Holstein cows were randomly assigned to receive 0, 50, 100, or 250 micrograms of the gonadotropin-releasing hormone product, Procystin when follicular cysts were diagnosed by palpation per rectum. Accurate reproductive records were maintained, and milk samples were collected at the time of diagnoses for assay for progesterone. An additional 101 cows were injected with only the 100 micrograms dose of Procystin when cysts were identified. Data showed that days from treatment to first observed estrus decreased with increasing doses of Procystin with no advantage of 250 micrograms over 100 micrograms. Days open and conception rates were similar among the treatment groups. Cows with less than 1 ng/ml progesterone in their milk at the time of treatment returned to estrus sooner than cows with progesterone concentrations greater than 1 ng/ml. In addition, gonadotropin-releasing hormone administered to those cows with low progesterone at the time of treatment led to significantly increased progesterone concentrations by 7 and 14 d posttreatment. We conclude that although Procystin administration hastened estrus of cows with ovarian cysts, breeding practices on the farms did not lead to an improvement in reproductive efficiency of the cows that possessed cysts.

Removal of glycosaminoglycans from bovine granulosa cells contributes to increased binding of hydrogen-3 heparin.

Granulosa cells from small or large bovine follicles were pretreated with enzymes that hydrolyze various glycosaminoglycans, and binding of [3H]-heparin to the granulosa was measured. Binding of [3H] heparin increased significantly after enzymatic pretreatments with chondroitinase ABC and fungal hyaluronidase, and similar results were obtained with granulosa from small and large follicles. No changes in binding of [3H] heparin were detected after hydrolyses with chondroitinase AC and heparinase in either follicle size. Heparitinase, which hydrolyzes heparan sulfate, led to a significant 50% increase in binding of [3H] heparin to granulosa from large follicles but was without effect in small follicles. These results suggest that the lower binding of [3H] heparin, which has been reported with follicular enlargement, may be due to heparan sulfate occupying or obstructing binding sites for heparin on granulosa from large follicles.

Glycosaminoglycans in follicular fluid from women undergoing in vitro fertilization and their relationship to cumulus expansion, fertilization, and development.

The glycosaminoglycans (GAGs) chondroitin sulfate (CS) and heparan sulfate (HS) were assayed in fluid from 178 individual follicles obtained from women after human menopausal gonadotropin ovulation induction for subsequent in vitro fertilization. CS and HS concentrations ranged from 0 to 3.9 and 0 to 18.6 mg/ml, respectively. The percentage of oocytes fertilized was significantly higher when CS concentrations ranged from 0.3 to 0.8 mg/ml (P less than 0.03). As HS levels increased, maturation scores by visual assessment of the egg-corona-cumulus cell complexes increased (P less than 0.05), but the percentage of oocytes fertilized was not affected. Cleavage rates of developing embryos were not related to the concentrations of GAGs.

Specific binding of [3H]heparin to bovine granulosa cell membranes.

Bovine granulosa cell membranes from small (SFM) and large (LFM) antral follicles were incubated with [3H]heparin, a commercial radioactively labeled glycosaminoglycan (GAG). Binding was specific, reversible, saturable, and dependent on time, pH, ionic strength and divalent cations. SFM exhibited different [3H]heparin binding characteristics compared to LFM. The addition of a physiological concentration of calcium (2 mM) yielded significant differences (P less than 0.02) in [3H]heparin binding for SFM (87 590 +/- 4206 dpm/10(6) cells) compared to LFM (55 230 +/- 2816 dpm/10(6) cells). SFM and LFM showed maximum [3H]heparin binding at pH 6.5 and pH 5.5, respectively. Increasing the ionic strength by addition of 0.07-2.0 M NaCl interfered with binding. Addition of unlabeled heparin (0.1-100 micrograms/ml) displaced [3H]heparin bound to SFM and LFM in a dose-dependent manner, as did dextran sulfate, a non-GAG sulfated branched polysaccharide. Commercial chondroitin sulfate ABC displaced the bound [3H]heparin only at doses between 50 and 500 mg/ml. GAGs purified from FF suppressed binding 39% at a concentration of 5.9 mg/ml. Photomicrographs of fluorescein-labeled heparin bound to granulosa cells showed localized areas of heparin binding to the cell surface. These experiments demonstrated that the GAG heparin specifically bound to bovine granulosa cell membranes, and that significant differences existed between the binding characteristics of SFM and LFM.

Relationships between bovine follicular fluid glycosaminoglycans and steroids.

The glycosaminoglycans chondroitin sulfate (CS) and heparan sulfate (HS) are present in follicular fluid. The present study evaluated estradiol (E), progesterone (P), CS, and HS concentrations from pools of 2633 small (less than 6 mm), 1702 medium (6-10 mm), and 491 large (greater than 10 mm) bovine follicles subdivided by relative E concentrations. E and P concentrations increased with follicle enlargement (P less than 0.05), but were inversely related within a follicle size. CS levels were reduced (P less than 0.05) in large (0.84 mg/ml) compared to small (1.18 mg/ml) and medium (1.36 mg/ml) follicles, while HS levels were decreased (P less than 0.05) in medium (0.34 mg/ml) and large (0.19 mg/ml) compared to small (1.10 mg/ml) follicles. Within a follicle size, CS and HS levels decreased significantly with increasing E. CS to HS ratios increased with follicle size and E concentration. The ratio of CS to HS was 4.4- and 1.6-fold higher, respectively, in medium and large follicular fluid samples classified healthy compared to atretic by their E content. These results support the idea that concentrations of glycosaminoglycans decrease with follicular maturation, and atretic follicles contain elevated levels of CS and HS.