The determination of 11 beta-hydroxyandrostenedione in human follicular fluid and plasma

The development of a chromatographic/immunoassay method is presented for the measurement of 11 beta-hydroxyandrostenedione (11 beta-OH-A4) in ovarian follicular fluid (FFL) and plasma from women undergoing embryo transfer for in vitro fertilization. This method incorporates high-performance liquid chromatography (HPLC) and permits the simultaneous measurement of other steroids from a single sample in order to assess the intraovarian environment. Authenticity of 11 beta-OH-A4 in follicular fluid was confirmed using selected ion monitoring (SIM) gas chromatography/mass spectrometry (GC/MS). Our results demonstrate a mean concentration of 18.6 nmol/l in follicular fluid compared with 3.2 nmol/l in plasma. The origin of 11 beta-OH-A4 in follicular fluid requires further investigation but these findings supports the hypothesis of ovarian 11 beta-hydroxylase activity on C19 steroids.     

Steroid 21-hydroxylase activity in equine ovarian follicles evidenced by isotope dilution-mass spectrometry

Steroid 21-hydroxylase activity of the microsome-enriched fraction of follicular linings from equine ovaries has been demonstrated by gas chromatography-mass spectrometry. The 21-hydroxylated metabolites were quantified by isotope dilution with deuterated analogues. The two most abundant potential substrates for follicular steroid 21-hydroxylase, progesterone (P) and 17-hydroxyprogesterone (17OHP), were converted respectively to 11-deoxycorticosterone (DOC) and 11-deoxycortisol with corresponding apparent specific activities of 308 and 24 pmol/mg protein/h and apparent Km values of 1.1 and 6.4 microM. Competitive inhibition of the P-to-DOC conversion was exerted by 17OHP and pregnenolone. Hence, the ovarian follicle of the mare is an extraadrenal site of preferential DOC biosynthesis by an enzyme having steroid 21-hydroxylase activity.     

Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11beta HSD) activity in follicular fluid from bovine and porcine large antral follicles and spontaneous ovarian cysts

In the ovary, cortisol is oxidized to cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). The present study investigated whether follicular fluid (FF) from large antral follicles and spontaneous ovarian cysts, isolated from bovine and porcine ovaries, contained modulators of 11betaHSD activity. Whereas FF from antral follicles had no significant effect over 1 h on NADP+-dependent 11betaHSD activity in rat kidney homogenates, enzyme activity was inhibited by FF from bovine and porcine ovarian cysts (80.5% +/- 2.3% and 72.8% +/- 3.4% of control, respectively). Following C18 reverse-phase chromatography, the hydrophilic fractions of FF from bovine and porcine antral follicles stimulated NADP+-dependent 11betaHSD activities (111.5% +/- 21.6% and 55.2% +/- 5.7% respectively). Hydrophobic compounds inhibited NADP+-dependent cortisol oxidation by 58.2% +/- 5.1% (bovine) and 45.7% +/- 2.0% (porcine). In both species, FF from ovarian cysts appeared to contain less of the hydrophilic stimuli to 11betaHSD activity and more of the hydrophobic inhibitors. The FF from antral follicles and ovarian cysts, and the C18 fractions thereof, had no significant effect on NAD+-dependent cortisol oxidation. The ovarian modulators of NADP+-dependent 11betaHSD activities did not coelute with cortisol, cortisone, estradiol, testosterone, progesterone, pregnenolone, and cholesterol. However, the 11betaHSD stimuli in porcine FF from both antral follicles and cysts coeluted with prostaglandin (PG) E2 and PGF2alpha. We conclude that large antral follicles and spontaneous ovarian cysts, in both the cow and the pig, contain ovarian modulators of the NADP+-dependent 11betaHSD activity. Moreover, FF from spontaneous ovarian cysts, because of decreased content of the 11betaHSD stimulus accompanied by increased content of the 11betaHSD inhibitors, exerts a net inhibitory effect on 11betaHSD activity.     

Carotene cleavage activity in bovine ovarian follicles

Carotene cleavage activity has been demonstrated in bovine ovarian follicles by incubating cell-free homogenates from granulosa cells with 15, 15'-(14)C-beta-carotene. Enzyme activity was highly dependent on the quality of follicles studied. Highest cleavage activity was found in large, preovulatory follicles. The correlation between follicular fluid concentration of vitamin A and the conversion rate obtained with the corresponding enzyme preparations was significantly positive. No correlation, however, could be shown between cleavage activity and beta-carotene, alpha-tocopherol or cholesterol. The only form of vitamin A in follicular fluid of cattle was found to be retinol. The results support the hypothesis that a local conversion of beta-carotene into vitamin A in follicular structures is responsible for the increase of the intrafollicular concentration of vitamin A during follicular development.     

A comparison of adrenal gland function in lactating dairy cows with or without ovarian follicular cysts

Two experiments were conducted to determine if adrenal secretion of steroids differed between cows that formed ovarian follicular cysts and normal cycling cows. In experiment 1, lactating Jersey and Holstein cows were diagnosed as having ovarian follicular cysts (follicle diameter >or=20 mm) by rectal palpation. Following diagnosis, ovaries were examined by transrectal ultrasonography three times weekly to detect subsequent ovulation (n=8) or new cyst formation (n=9). Venous blood samples were collected daily to quantify circulating concentrations of cortisol and progesterone. The average concentration of cortisol during the 10-day period prior to ovulation was not different from the concentration prior to the formation of a new cyst. In experiment 2, secretion of cortisol and progesterone was examined in cows with ovarian follicular cysts (n=4) and cyclic, control cows in the follicular phase of the estrous cycle (n=4). An adrenocorticotropic hormone (ACTH) challenge was administered to cystic cows 4-7 days after new cyst formation and to cyclic cows in the follicular phase of the cycle (36 h after induction of luteolysis). Jugular venous blood samples were collected at -60, -30, 0, +10, +20, +30, +60, +90, +120, +180, +240, +300 and +360 minutes relative to ACTH administration. A rapid increase in both cortisol and progesterone was observed immediately following administration of ACTH in each treatment group. Peak concentrations of both steroids were achieved within 60 minutes after administration of ACTH. Concentrations of cortisol and progesterone did not differ between cystic and cyclic cows. In summary, no differences in adrenal function were detected between normal cycling cows and cows with ovarian follicular cysts.     

Biochemical and hormonal composition of follicular cysts in water buffalo (Bubalus bubalis)

The objective of this study was to examine the follicular fluid biochemical and hormonal changes associated with ovarian follicular cysts in buffalo. Follicular fluid was aspirated from eight cysts and eight preovulatory follicles, and subjected to biochemical and hormonal analyses. Cysts were characterized by a greater (P<0.01) concentration of nitric oxide and lesser concentrations of ascorbic acid and glucose than that of preovulatory follicles (P<0.01 and P<0.05, respectively). Furthermore, follicular cysts had greater concentrations of progesterone (P<0.001), triiodothyronine (T(3)) and cortisol (P<0.05) and lesser concentrations of insulin (P<0.001) than preovulatory follicles. The results indicate follicular cysts in buffalo have an altered biochemical and hormonal composition. The alterations include increases in nitric oxide, progesterone, cortisol and T(3) concentrations with a concurrent reduction in ascorbic acid, insulin and glucose concentrations. The study suggests that greater progesterone concentrations possibly inhibit the onset of LH surge resulting in formation of follicular cysts in buffalo. In addition, it implies the plausible role of intra-ovarian regulators such as nitric oxide, ascorbic acid and insulin in development of the condition.     

Follicular fluid adrenomedullin concentrations in spontaneous and stimulated cycles: relationship to ovarian function and endothelin-1 and nitric oxide

The objective of this study was to determine concentration of adrenomedullin (AM) in follicular fluid and whether a correlation exists between AM and nitric oxide (NO) or endothelin-1 (ET-1) levels in follicular fluid, serum 17beta-estradiol or other parameters of ovarian function in spontaneous and gonadotrophin stimulated ovarian cycles. Follicular fluid samples were obtained at oocyte retrieval from 50 women who underwent an in vitro fertilization (IVF) program: 40 undergoing ovarian hyperstimulation with recombinant FSH and 10 had spontaneous ovarian cycles. AM, ET-1, and NO were detected in all of the follicular fluid samples and their concentrations were similar in spontaneous and stimulated cycles. In patients undergoing ovarian stimulation, follicular fluid AM levels correlated with serum 17beta-estradiol concentration. No correlation was found between follicular AM concentration and parameters of ovarian function. Similarly, no relationship was observed between ET-1, NO, and AM follicular fluid concentrations in either spontaneous or stimulated cycles.This study suggests a possible regulatory effect of the sexual hormones on AM production by the ovary during the ovulatory process. The site of AM secretion and its function (if any), however, remain to be established.     

Sex steroid changes in porcine cystic ovarian disease

Ten sex steroids were measured in the peripheral serum and ovarian follicular fluid of female pigs with or without cystic ovarian disease. In general, progestin, especially progesterone, accumulated excessively in the fluid contained in cystic compared with normal follicles. Nonluteinized cystic follicles contained up to four times the progesterone concentration found in large normal preovulatory follicles. Levels of this steroid increased with luteinization of cystic follicles to as much as 10 times those found in large preovulatory follicles. In contrast, the concentration of follicular fluid androgens and estrogens in cystic follicles were, at best, barely detectable (5 to 10 pg/ml). These results are indicative of a steroidogenic blockade in the conversion of C21 progestin to C19 androgens and C18 estrogens in the cystic follicles. In spite of an enormous accumulation of follicular progestin and subnormal concentration of androgens and estrogens, circulating levels of these hormones in pigs bearing cystic ovaries were in the normal range for cycling sows. Clearly, the hormonal abnormalities in the cystic follicles are not reflected in the serum profiles of these steroids.     

Presence of an aromatase inhibitor, possibly heat shock protein 90, in dominant follicles of cattle.

In cattle, it has been suggested that follicular fluid has direct modulatory effects on follicular growth and maturation. In the first part of this study, an in vitro test using aromatase activity of follicular wall fragments as an end point was validated for cattle follicles and was used to test whether follicular fluid (from dominant or non-dominant follicles) modulates aromatase activity. Fluid from dominant follicles at a concentration of 24 or 12% (obtained during the luteal and follicular phases, respectively) significantly inhibited aromatase activity. Inhibitory activity was low or absent in fluid from non-dominant follicles. FSH-stimulated aromatase activity was also reduced by fluid from dominant follicles, but not to a greater extent than in basal conditions. Finally, charcoal-treated fluid from dominant follicles retained its inhibitory activity. In contrast, ovarian venous serum draining a dominant follicle had no activity at the three concentrations tested (6, 12 and 24%). In the second part of the study, identification of the compounds involved in this modulatory activity was attempted using SDS-PAGE. Comparison of the fluorographs from de novo synthesized proteins stored in follicular fluid (inhibitory medium) with those secreted in incubation medium (inactive medium) demonstrated that one protein (90 kDa, pI 5.8) was significantly (P < 0.05) more abundant in fluid from dominant follicles (2.0 +/- 0.09%) than in the culture medium (1.3 +/- 0.1% of the total proteins). This protein had characteristics similar to those of heat shock protein 90 (hsp 90). Therefore, in the final part of the study, the presence of hsp 90 in ovarian cells and follicular fluid was investigated using immunohistochemistry and western blot analysis. After immunohistochemistry, a positive signal was detected mainly in the granulosa cells of larger follicles and to a smaller extent in thecal cells and oocytes. Western blot analysis also demonstrated the presence of hsp 90 in follicular wall fragments and fluid. When blotting was achieved on a sample of follicular fluid resolved by two-dimensional PAGE, the spot detected had a similar location to that at 90 kDa and pI 5.8. Addition of purified hsp 90 to bovine follicles in vitro depressed aromatase activity by altering the K(m) value (and possibly the Vmax value) of the enzyme. It is proposed that hsp 90 is a functional regulator of follicular maturation through its action on aromatase.     

Induction of maturation of Atlantic croaker oocytes by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one in vitro: consideration of some biological and experimental variables

We characterized the in vitro control of germinal vesicle breakdown (GVBD) by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) in intact ovarian follicles of gonadotropin-primed Atlantic croaker. 20 beta-S-induced GVBD was determined in relation to ovarian (oocyte) morphology, duration of incubation, steroid metabolism, and interaction with other steroids. The rate of GVBD in vitro in the absence of exogenous steroid was positively correlated with initial stage of ovarian morphological development. Maximal responsiveness to 20 beta-S was seen in ovaries with oocytes showing the first signs of morphological maturation. Dose-response experiments with 20 beta-S and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P) over a range of incubation times yielded similar results for both steroids, suggesting that conversion of 17 alpha,20 beta-P to 20 beta-S is not required for 17 alpha,20 beta-P-induced GVBD. The ED50 of these steroids markedly decreased with increasing incubation times. Comparisons between patterns of follicular transformation of various radiolabelled steroids to 20 beta-S and their respective activities (using unlabelled steroids) in the GVBD bioassay suggested that, in addition to 17 alpha,20 beta-P, progesterone has some intrinsic maturational activity. However, the maturational effects of 11-deoxycortisol and pregnenolone may be explained by their conversion to 20 beta-S. For the first time in any vertebrate, we showed that the proposed maturation-inducing steroid (20 beta-S) is not significantly transformed to any extractable, potentially active metabolite by intact, maturing ovarian follicles. These findings strongly suggest that 20 beta-S is the terminal product of the MIS biosynthetic pathway in Atlantic croaker ovaries. Estradiol had no acute effects on 20 beta-S-induced GVBD. However, testosterone decreased and cortisol augmented the maturational activity of 20 beta-S. Excess progesterone reduced the activity of a maximally effective dose of 20 beta-S, but pregnenolone was without effect. The effects of these steroids on 20 beta-S-induced GVBD are discussed in relation to their possible interactions with 20 beta-S at the MIS receptor level.     

Stimulatory and Inhibitory Effects of Human Follicular Fluid on Amphibian Oocyte Maturation and Ovulation hi vitro

Follicular fluid was collected from individual human ovarian follicles and its effects, alone or in combination with frog pituitary homogenate (FPH), on oocyte maturation and ovulation were assessed following incubation with amphibian ovarian follicles in vitro. Oocyte maturation, with little or no concomitant ovulation, was induced by variable amounts of follicular fluid. Some of the individual follicular fluid samples were very active in inducing oocyte maturation, whereas others were inactive. Frog pituitary homogenates exhibited biologic activity (induced oocyte maturation and ovulation) when incubated in the presence of most follicular fluid samples. However, follicular fluid samples from two individuals inhibited ovulation but not maturation in FPH-treated follicles. These results demonstrate that amphibian follicles remain viable and undergo a number of physiologic changes in the presence of unfractionated human follicular fluid. Under appropriate conditions both stimulatory and inhibitory effects of follicular fluid were observed. These data suggest that amphibian ovarian follicles may provide a simple and independent means for detecting and assaying a number of biologic activities present in follicular fluid obtained from single human and other mammalian ovarian follicles. Such results may provide the basis for dissociating endocrine and cellular interactions which occur during normal and abnormal follicular differentiation.     

Inhibition of gonadotropin sensitive adenylate cyclase by ovarian follicular fluid.

Follicular fluid obtained from medium or large bovine ovarian follicles inhibited ovarian luteinizing hormone/human chorionic gonadotropin sensitive adenylate cyclase in a dose-dependent manner (I₅₀ = 3 mg follicular fluid protein/ml). The inhibitory activity was excluded by Sephadex G-10 and was fully retained following treatment with charcoal. Fluoride-stimulated enzyme activity was not inhibited. Binding of ¹²⁵I human chorionic gonadotropin to ovarian plasma membranes was only slightly reduced by the follicular fluid. The post-microsomal supernatant of homogenates from ovaries of immature (27-day-old) rats collected 24–36 h after treatment with 15 i.u. of pregnant mare serum gonadotropin also inhibited luteinizing hormone-sensitive adenylate cyclase. The extent of this inhibition seemed to decline with follicular maturation. The possibility is raised that ovarian sulfated glycosaminoglycans are responsible for the observed inhibition of adenylate cyclase.     

Relationship between concentrations of cortisol in ovarian follicular fluid and various biochemical markers of follicular differentiation in cyclic and anovulatory cattle

Concentrations of cortisol were determined in pooled fluid of small (less than 10 mm) and large (greater than or equal to 10 mm) follicles of cyclic cattle (Exp. 1), and in fluid of the largest follicle of 17 post-partum anovulatory cows (Exp. 2). In Exp. 1, concentrations of cortisol in small follicles were greater (P less than 0.05) than in large follicles (14.7 versus 13.2 ng/ml), and varied significantly with stages of the cycle; small and large follicles had the highest cortisol concentration during the early luteal phase of the cycle. Large follicles had 2-fold greater concentrations of oestradiol than did small follicles, whereas small follicles had 2-fold greater concentrations of androstenedione than did large follicles. Across pools of follicular fluid, cortisol concentrations were correlated only to androstenedione concentrations (r = 0.65, P = 0.07). In Exp. 2, concentrations of cortisol did not significantly differ between oestrogen-active (oestradiol greater than progesterone in follicular fluid) and oestrogen-inactive (progesterone greater than oestradiol) follicles, although oestrogen-active follicles had a 24-fold greater concentration of oestradiol than did oestrogen-inactive follicles. Cortisol concentrations were correlated to hCG binding capacity of thecal cells (r = -0.35, P = 0.08) and to follicular diameter (r = 0.45, P less than 0.05). These results suggest that normally fluctuating concentrations of cortisol in follicular fluid of cattle play little or no active role in follicular differentiation in vivo.     

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.     

Type 2 11beta-hydroxysteroid dehydrogenase activity in human ovarian cancer

In the ovary cortisol-cortisone inter-conversion is catalyzed by the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD). Its role in carcinomas of human ovary is unknown. The majority of ovarian cancers are derived from ovarian surface epithelium and the inflammation caused by successive ovulation seems to a play a role in the development of cancer. Cortisol is known to act as anti-inflammatory agent and its metabolism by type 1 and type 11beta-HSD may control the inflammatory action by cortisol in ovary. We undertook this study to investigate type 2 11beta-HSD activity which functions exclusively oxidative direction, in normal ovarian tissue compared to ovarian epithelial cancer. Ovarian tissue was obtained from patients undergoing hysterectomy for both benign and malignant disease. Tissue was placed immediately on dry ice and subsequently transferred to a freezer where they were maintained at -70 degrees C. NAD dependent 11beta-HSD activity was then determined in this tissue. T-test was performed to determine statistical significance. Mean type 2 enzyme activity was 0.87 +/- 1.65 pmol/min g tissue in normal ovarian tissue versus a mean enzyme activity of 2.96 +/- 1.37 pmol/mim g tissue in from cancer specimens. This difference was statistically significant with a p-value of 0.03. Type 2 1beta-HSD activity in ovarian cancer specimens was significantly higher than enzyme activity measured in normal post-menopausal ovarian tissue. Decreased cortisol levels due type 2 1beta-HSD activity may play a role neoplastic transformation as well as tumor proliferation in ovarian cancer by eliminating anti-inflammatory action of cortisol.     

Enzyme immunoassay for cortisol in serum using cortisol 21-amine

Cortisol 21-amine (21-amino-11β,17-dihydroxy-4-pregnene-3,20-dione) was prepared and an enzyme immunoassay for cortisol in serum was established using cortisol 21-amine conjugated with alkaline phosphatase. The minimal amount of cortisol detected was 1ng/tube and the measurable range was from 1 to 80 μg/d1, using 10 μ1 of serum sample. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision. The values correlated well with those obtained by radioimmunoassay. This enzyme Immunoassay is applicable to the routine determination of serum cortisol in any clinical laboratory. Cortisol 21-amine was found to be a useful derivative for preparing cortisol-enzyme conjugate in enzyme immunoassay.     

Bovine ovarian follicular fluid vitronectin content is influenced by the follicle size

Vitronectin was quantified in the follicular fluid aspirated from bovine follicles with diameters of 3 to 15 mm (as determined by ultrasonography) using a specific enzyme-linked immunosorbent assay (ELISA) validated for bovine vitronectin. The primary antibody was raised in rabbit against vitronectin purified from bovine plasma. Vitronectin quantified in serial dilutions of bovine plasma and ovarian follicular fluid was highly correlated with the volume of each sample assayed. In addition, known amounts of purified bovine vitronectin added to samples of plasma or follicular fluid were accurately recovered. Follicular fluid concentrations of vitronectin were positively correlated with the follicle diameter (r = 0.8; P < 0.01). These data indicate that bovine follicular fluid concentration of vitronectin is influenced by the stage of follicular development.     

Dexamethasone-suppressible hypercorticosteronism in two 46,XX subjects with ambiguous genitalia and ovarian cysts. Partial defect of 17 alpha-hydroxylase or 17-20-desmolase

The paradoxical association of female pseudohermaphroditism and androgen deficiency was observed in two 46,XX subjects with high corticosterone plasma levels. Subject 1 has been declared a boy due to clitoris enlargement; she had no vagina and uterus. Subject 2 had ambiguous external genitalia. In both, at age 27 and 17 years, fusion of outer labia, impuberism, ovarian cysts, and histologically normal ovarian tissue were observed. Blood pressure was normal. Basal cortisol levels were normal but unresponsive to ACTH. Progesterone levels were 40 and 62 ng/ml and rose after ACTH (50 and 79 ng/ml). 17-hydroxyprogesterone levels were 25 and 21 ng/ml and did not rise after ACTH. Corticosterone levels were 70 and 92 ng/ml and rose after ACTH (110 and 180 ng/ml). All three steroids were suppressed by dexamethasone. Androgen and estrogen levels were at or below the lower limit for normal women. The sex steroid levels obtained by radioimmunoassay in plasma and a follicular cyst fluid were confirmed by isotope dilution-mass spectrometry. We suggest that the sexual ambiguousness resulted from an excessive production of gestagenic steroids during fetal life, and that the enzyme defect is either a partial 17 alpha-hydroxylase defect combined with a peripheral production of 17-hydroxyprogesterone, or else a partial 17-20-desmolase defect with a secondary 21-hydroxylase defect limited to the cortisol pathway.     

Presence of bacteria in porcine follicular fluid and their ability to generate an inhibitor of follicle-stimulating hormone binding to receptor

Follicular fluid obtained from porcine ovaries collected at slaughter and distributed by the National Institutes of Health was contaminated by bacteria which appeared to be of intestinal origin. This follicular fluid showed increased follicle-stimulating hormone binding inhibition (FSH-BI) activity following incubation under conditions which facilitated bacterial growth. No such increase in FSH-BI activity was observed following incubation of follicular fluid from which bacteria were removed by repeated filtration. Our data suggest that bacteria found in the follicular fluid were capable of generating a substance with FSH-BI activity. This substance has an apparent molecular weight greater than 6000, based on membrane diafiltration studies. The possible presence of bacteria in follicular fluid and their ability to generate a substance which interferes with FSH binding to receptor should be considered in studies on factors in follicular fluid that are considered to regulate ovarian function or development.     

Alterations in follicular fluid estradiol, progesterone and insulin concentrations during ovarian acyclicity in water buffalo (Bubalus bubalis)

Ovarian acyclicity is one of the most important causes of infertility in water buffalo. Recent studies have indicated alterations in the composition of follicular fluid during the condition. The aim of this study was to determine the changes in follicular fluid concentrations of estradiol, progesterone and insulin during ovarian acyclicity in water buffalo. Ovaries were collected from 50 acyclic and 95 cyclic (control) buffaloes and follicular fluid was aspirated from small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized follicles. Estradiol concentration was lower (P<0.0001) in acyclic (1.4 ± 0.09 ng/ml) than in cyclic (3.3 ± 0.18 ng/ml) buffaloes. Regardless of the ovarian cyclic status, there was an increase (P<0.01) in estradiol concentration with the increase in follicle size; the mean concentrations were 2.4 ± 0.16 ng/ml, 2.8 ± 0.29 ng/ml and 3.5 ± 0.41 ng/ml in small, medium and large follicles, respectively. A higher (P<0.001) progesterone concentration was recorded in acyclic (24.3 ± 2.61 ng/ml) compared to the cyclic (7.6 ± 0.79 ng/ml) group. Furthermore, acyclic buffaloes had a lower (P<0.05) concentration of insulin in the follicular fluid than that of cyclic buffaloes (15.2 ± 1.55 μIU/ml versus 25.9 ± 2.78 μIU/ml, respectively). In conclusion, acyclic buffaloes have lower concentrations of estradiol and insulin concurrent with higher concentrations of progesterone in the follicular fluid. These hormonal changes in the follicular microenvironment are possibly a manifestation of the disturbances in the normal follicular development leading to anovulation and anestrus in acyclic buffaloes.