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.     

Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period

Background  

Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation.     

Effect of homologous preovulatory follicular fluid on in vitro maturation of equine cumulus-oocyte complexes

The objective of this study was to test the hypothesis that incubating equine cumulus-oocyte complexes (COCs) in medium containing 50% or 100% homologous preovulatory follicular fluid would improve cumulus expansion and nuclear maturation. Oocytes were incubated in one of three media: 1) supplemented TCM-199 (control), 2) 50% (v/v) follicular fluid in control medium or 3) 100% follicular fluid. Cumulus expansion was evaluated subjectively, and nuclear maturation was evaluated by staining oocytes with Hoechst 33258. The hypothesis that incubating COCs in medium containing follicular fluid would improve cumulus expansion was supported. More (P < 0.05) compact COCs incubated in 50% or 100% follicular fluid developed a moderately to completely expanded cumulus after 24 and 36 h of incubation and more (P < 0.05) expanded COCs incubated in 100% follicular fluid developed a moderately to completely expanded cumulus after 36 h of incubation compared to control medium. The hypothesis that incubating COCs in medium containing follicular fluid would improve nuclear maturation was not supported. Although more (P < 0.05) compact COCs incubated in 50% follicular fluid reached polar body-stage compared to those in control medium, the nuclear maturation rate in the control medium was lower than it was when the same medium was used in a preliminary experiment (described in main text); therefore, the apparent superiority of 50% follicular fluid must be interpreted cautiously. Based on these results, future studies are warranted to further address the value of adding preovulatory follicular fluid to equine IVM culture systems.     

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.     

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.     

Nature of the rabbit acrosome reaction-inducing activity of follicular fluid.

Follicular fluid samples from rabbits, cats, pigs, women and cows had acrosome reaction-inducing activity (ARIA) on rabbit spermatozoa as determined by differential staining after incubation with these fluids. Activity was retained after dialysis and at least 50% was found to be labile when heated to 56 degrees C for as little as 20 min. The induction of the acrosome reaction by bovine follicular fluid showed a dependence on the concentration of follicular fluid and spermatozoa and on calcium ions, and had a pH optimum of approximately 8. Enzyme treatments showed that proteases destroyed the ARIA and this activity was completely blocked by treatment of bovine follicular fluid with goat anti-bovine antiserum. Electron microscope observations indicated the similarity of the reactions observed to that occurring in vivo. It is concluded that the rabbit acrosome reaction inducing-activity of bovine follicular fluid is a serum component(s), probably a protein(s) of high molecular weight.     

Hypocoagulable state of human preovulatory ovarian follicular fluid: role of sulfated proteoglycan and tissue factor pathway inhibitor in the fluid

Ovulation accompanied by tissue damage can cause an increase in the level of tissue factor (TF) in the follicular fluid, triggering the extrinsic coagulation pathway. However, follicular fluid must block fibrin formation and maintain fluidity until the release of the oocyte at ovulation. The combination of sulfated proteoglycan, antithrombin, and TF pathway inhibitor (TFPI) appears to play a critical role in the hypocoagulability of human follicular fluid. When compared with plasma, folicular fluid differs markedly in the levels of a number of important coagulation proteins. Principal among these are 15-fold, 13-fold, and 3.7-fold increases in free TFPI, thrombin-antithrombin complex, and TF, respectively. The excessively prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) of human ovarian follicular fluid appear to be primarily due to high concentrations of sulfated proteoglycans, which accelerate the inactivation of thrombin and the anti-Xa activity of TFPI. Thus, heparitinase treatment shortened the clotting times of follicular fluid and reduced the inhibition of thrombin by the proteoglycan fraction combined with a fraction containing antithrombin. The remaining prolongation of APTT and PT may be caused by high levels of free TFPI in follicular fluid, which were confirmed by Northern blotting analysis, demonstrating TFPI mRNA expression by granulosa cells.     

Changes in follicular fluid concentrations of steroids, the pattern of steroid secretion and aromatization capability of incubated preovulatory follicular wall of rats

Female Wistar rats exhibiting a regular 4-day oestrous cycle were included in this study. They were killed on the day of pro-oestrus at 11.00, 14.00, 16.00, 2.00, 22.00 and 24.00 h. The isolated preovulatory follicles were placed individually on a small triple disc of filter paper. They were cut in half and the flowing out follicular fluid (FF) was allowed to soak into the underlying filter paper disc, which was subsequently placed at the bottom of incubation chamber. It was rinsed with the incubation medium and this medium, containing FF, was collected. The remaining follicular wall was incubated for 3h. Some of the incubation media were supplemented with testosterone (T). The steroid content was estimated by radioimmunoassay. The secretion of estradiol (E) by follicular wall was high until 20.00 h while that of androgens until 22.00 h, and both declined thereafter. Relatively low progesterone (P) release rose sharply at 22.00 h and this hormone became the main steroid secreted at 24.00 h. The addition of T always enhanced the release of estradiol. Follicular fluid contained very little estradiol, while prevailing steroids were androgens. Decrease of androgen concentrations was found only at 24.00 h. Progesterone level, much lower than that of androgens, started to rise at 20.00 h and P was the main steroid present in FF at 24.00 h. The present results suggest an existence of selective passage of steroids into FF and show a presence of androgenic milieu surrounding cumulus oophorus in preovulatory FF. The results also indicate that the aromatase activity is sustained until ovulation and stress the role of the P rise observed in the secretion rate of follicular wall and FF content just before ovulation.     

Changes in biochemical composition of follicular fluid during reproductive acyclicity in water buffalo (Bubalus bubalis)

This study describes the changes in biochemical composition of follicular fluid during reproductive acyclicity in buffalo. A total of 73 pairs of ovaries collected from 26 reproductively acyclic and 47 reproductively cyclic buffaloes were used in the investigation. Ovarian follicles were classified into small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized categories depending upon their diameter. Follicular fluid was aspirated, processed and assayed for glucose, cholesterol, total protein, acid phosphatase and alkaline phosphatase. Glucose concentration was lesser in reproductively acyclic compared to cyclic buffaloes (19.3 ± 2.59 mg/dl compared to 32.6 ± 2.60 mg/dl; P<0.05), mainly due to difference in concentration between small sized follicles (12.4 ± 2.59 mg/dl compared to 28.0 ± 3.32 mg/dl; P<0.05). Cholesterol concentration was also lesser in reproductively acyclic compared to cyclic buffaloes (32.2 ± 2.14 mg/dl compared to 35.5 ± 2.16 mg/dl; P<0.05) and this was related to the lesser concentration found in large follicles (13.8 ± 3.45 mg/dl compared to 37.2 ± 4.10mg/dl; P<0.001). Total protein and acid phosphatase levels were not affected by either the reproductive cyclicity status or the follicular size (4.9 ± 1.07 g/dl to 6.0 ± 0.28 g/dl and 1.2 ± 0.17 U/dl to 2.5 ± 1.22 U/dl, respectively). An increased alkaline phosphatase activity was, however, observed in reproductively acyclic compared to cyclic buffaloes (27.5 ± 3.08 U/dl compared to 14.0 ± 1.09 U/dl; P<0.0001). In conclusion, results of the present study indicate an alteration in the biochemical composition of follicular fluid during reproductive acyclicity in buffalo. The findings provide further support to the notion that poor nutrition is an important factor triggering reproductive acyclicity in buffalo.     

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)     

Changes in follicular blood flow and nitric oxide levels in follicular fluid during follicular deviation in cows

Two experiments were conducted to test the hypothesis that there are dynamic changes in follicular blood flow during follicular deviation and that nitric oxide (NO) in follicular fluid (FF) plays a role in regulation of follicular blood flow. In Experiment I, follicular blood flow of the two largest follicles was monitored by using Power Doppler ultrasonography during follicular deviation in sixteen follicular waves during eight estrous cycles in eight cows. Blood flow did not differ (P>0.05) between the dominant follicle (DF) and the largest subordinate follicle (SF) until the beginning of the deviation of the follicular size, but was higher (P<0.05) in DF than in the largest SF one and two days after the beginning of diameter deviation in ovulatory (n=5) and atretic (n=11) waves; respectively. In Experiment II, FF was aspirated from DF and the largest SF on the day of diameter deviation (DF, n=6; SF, n=6) and two days later (DF, n=12; SF, n=9). Nitric oxide did not differ (P>0.05) between DF and the largest SF on the day of diameter deviation but, one or two days after observed diameter deviation NO concentrations were lower (P<0.01) in DF compared to the largest SF. On the day of diameter deviation and two days later E2 levels in FF were higher (P<0.01) in DF than in the largest SF. P4 concentrations in FF were higher (P<0.05) in DF than in the largest SF on the day of diameter deviation, but did not (P>0.05) differ two days later. E2/P4 ratio in FF was the same (P>0.05) in DF and the largest SF on the day of diameter deviation, but was higher (P<0.01) in DF than in the largest SF one or two days later. In conclusion, area of follicular blood flow of DF and the largest SF increased in parallel with follicular size during follicular deviation. Furthermore, there were relationships between changes in follicular blood flow, NO concentrations and E2/P4 ratio in FF following the beginning of diameter deviation in cattle.     

Qualitative and quantitative changes in protein synthesis of bovine follicular cells during the preovulatory period.

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.     

The effects of porcine follicular fluid inhibin on gonadotropin secretion in the rabbit

Porcine follicular fluid (pff), treated with charcoal to remove steroids, was used to determine whether inhibin is active in the laboratory rabbit. When pff (5 ml/4 kg body weight) was injected (ip) into does that had been castrated 2 weeks earlier, there was a significant decline in blood follicle-stimulating hormone (FSH) levels; the decline lasted for 8-12 h. Blood levels of luteinizing hormone (LH) were suppressed, but only briefly at 3 h after injection. In other experiments, intact does which had been injected with pff 9 h and 10 min before receiving a single, i.v. injection of luteinizing hormone-releasing hormone (LHRH) (10 micrograms/kg body weight) showed a sharp reduction in the concentration of LH in the blood samples collected 15, 30 and 60 min after LHRH administration. Secretion of FSH responded poorly to LHRH stimulation, and pff had little suppressive action on blood levels. Having established that the pff preparation had inhibin activity, its action on the postovulatory surge of FSH secretion was next examined. This release of FSH, which occurs 6 to 36 h after ovulation, has been hypothesized to be required for the establishment of pregnancy by stimulating the growth of the ovarian follicles supplying the luteotropic estradiol. To test this hypothesis, pff was injected into rabbits every 8 h for the first 5 days of pregnancy and found to block the postovulatory FSH surge. The patterns of secretion of LH and progesterone in the same pff-injected animals were, however, not altered from normal pregnancy patterns by pff.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian epidermal growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement

Numerous data indicate that epidermal growth factor has important effects on cultured granulosa cells. However, most of the few attempts to detect epidermal growth factor in ovarian tissue have been unrevealing. In this study, ovarian epidermal growth factor-like activity was easily detected by a radioreceptor assay based on the A431 cell line but not by an immunoassay for mouse epidermal growth factor. The concentration of this activity in follicular fluid from small porcine ovarian follicles was higher than that in fluid from medium or large follicles or serum (p less than 0.01), but lower than that in salivary gland extracts. Receptor-active epidermal growth factor-like peptides could function as local ovarian regulators.     

Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage

Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia.