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Elevated inhibin concentration in the follicular fluid of dairy cows with chronic cystic ovarian disease
Theriogenology
40:809-818,1993
ELEVATED INHIBIN CONCENTRATION IN THE FOLLlCUlAFl FLUID OF DAIRY COWS WlTH CHRONIC CYSTIC OVAMAN DISEASE
S. Roberge,la J.L. Brown* and J.J. Reeves’
’ Department of Animal Science, Washington State University Pullman WA 991646332 *Conservation and Research Center, Front Royal VA 22636
Received for publication: hxmnber 15, 2992 Accepted: June 8, 199.3 ABSTRACT This study compared serum and follicular fluid inhibin and gonadotropin profiles between chronic cystic ovarian diseased (CCOD) and normal cyclic dairy cows. Blood samples and follicular fluid were collected from CCOD cows (n=15) and cyclic cows in the follicular phase of the estrous cycle (control, n=6) and analyzed for inhibin, follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations. There was a significant increase in inhibin and a decrease in FSH and LH concentrations in the follicular fluid of CCOD cows compared with those of cyclic cows (P e 0.05). Mean serum inhibin, FSH and LH concentrations between CCOD and cyclic cows were not different (P > 0.05) however, there was a tendency for serum inhibin to be higher and FSH to be lower in CCOD cows compared to cyclic animals (P e 0.1). The FSH pulse frequency also was lower in CCOD cows than in cyclic cows (P < 0.05). These data suggest that increased production of inhibin from cystic follicles of CCOD cows alters pituitary FSH secretion and subsequently reduces the concentration of FSH in follicular fluid. As a result, decreased FSH stimulation at the ovarian level could ultimately lead to the reduction in follicular LH and FSH receptor concentrations, resuiting in abnormal follicular steroidogenesis in CCOD dairy cows.
Key words: inhibin, FSH, cyst, follicular fluid, cystic cow Acknowledgments Scientific paper no. 6656. College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA 99164. ‘Present address and correspondence: Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, CANADA Ni G 2Wl. FAX: (519) 767-6573.
Copyright Q 1993 Butterworth-Heinemann
Theriogenology
810 INTRODUCTION
Ovarian cysts in dairy cows are generally defined as anovulatory follicular structures, at least 2.5 cm in diameter, that persist for 10 days or more in the absence of a corpus luteum (CL; 1,2). Approximately 80% of cows with cystic ovarian disease (COD) respond to treatment with human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRH; 3-9). These treatments induce the release of endogenous luteinizing hormone (LH) and cause luteinization of the cystic structure and a reestablishment of ovarian cyclicity within 21 to 24 days of treatment (4-8). However, 29% of COD cows do not respond to hormone treatment and are referred to as chronic cystic-ovarian diseased (CCOD) cows (5,8,8). Some studies have reported that the cystic condition is apparently not related to a pituitary dysfunction, since neither serum LH or FSH secretion, nor pituitary LHRH receptor concentrations are altered in cystic cows compared with that of cyclic cows (8, 9-l 1). Instead, it has been suggested that the inability of CCOD cows to respond to hormonal treatment is due to a specific dysfunction at the ovarian level (12). In that study, a significant decrease in both LH and FSH receptors in the ovarian wall and follicular granulosa cells was observed in CCOD cows compared with that of cyclic cows. There was also a higher ratio of androgens to estrogens in the fluid of cystic follicles compared to that of non atretric follicles of cyclic cows (12). Reduced follicular aromatase activity in cystic follicles, resulting in reduced estrogen production, has also been reported (2,13-l 4). In addition to producing steroids, granulosa cells also produce a glycoprotein hormone, inhibin, which plays an important role in suppressing pituitary FSH secretion both in vivo and in vitro (15). lnhibin also directly inhibits FSH-induced granulosa cell estrogen production (15). It has been demonstrated that there is presence of large amounts of inhibin in bovine follicular fluid (18). In polycystic ovarian diseased women, higher concentrations of inhibin in follicular fluid of cystic follicles have been observed than in normal follicles (17-l 9). Likewise, high concentrations of inhibin in follicular fluid from rat cystic follicles were accompanied by a decrease in serum FSH secretion (29). There is, as yet, no report on the role of inhibin in CCOD dairy cows. Therefore, the objectives of this research project were to compare serum and follicular fluid inhibin, FSH and LH profiles between CCOD and normal cyclic cows. MATERIALS AND METHODS Animals Fifteen lactating Holstein cows, diagnosed as cystic by veterinarians using palpation per rectum, were used in this study. These were termed CCOD cows because they had been treated an average of 8 times with hCG or LHRH without response and had been in a chronic cystic condition for an average of 10 months. All cows were lactating and were milked twice daily until termination of the study. Six noncystic, cyclic Holstein control cows in the luteal phase of the estrous cycle were injected with 35 to 50 mg of PGFsaband were slaughtered approximately 48 to 50 hours after injection, to ensure that they were in the follicular phase at the time of slaughter. Samples from 7 CCOD cows and from all control cows were utilized in a previous study (12). Samples from an additional 8 CCOD cows were collected within the same year of the above study. b Lutalyse, Upjohn Co., Kalamazoo,
MI.
Theriogenolog
y
Blood Sampling and Follicular Fluid Collection All cows were fitted with jugular catheters the day before slaughter and blood samples were collected every 15 minutes for 4 hours. Approximately 12 hours after collection, all the animals were slaughtered, and the ovaries were removed, placed on ice, and examined for follicular and luteal development. Ovaries from the CCOD cows contained singular cysts that exceeded 2.5 cm in diameter, and had no functional CL present. Control cows were considered to be follicular if the ovary contained a follicle 1.5 to 2.0 cm in diameter. Follicular fluid from control and cystic follicles was aspirated using a syringe fitted with a 2O-gauge needle. Serum and follicular fluid were stored at -20°C until assayed for inhibin, FSH and LH concentrations. Radioimmunoassays Serum and follicular fluid concentrations of FSH were determined by a double antibody radioimmunoassay (FM) as described previously using USDA-bFSH-B-1 asthe hormone standard (21). Intra- and inter-assay coefficients of variation (CV) were 8.6 and 11.2%, respectively. Assay sensitivity was 7.5 ng/ml. Concentrations of LH were determined in a single MA as described previously using NIH-LH-B7 as the hormone standard (22). Intra-assay CV was 5%. The sensitivity of the assay was 0.3 ng/ml. Concentrations of inhibin were determined by a RlA previously described (23), using antiserum raised against a synthetic porcine a(l-30) inhibin fragment (JLB #492) and the same synthetic a(1-30) fragment as the labeled hormone. Displacement of ‘“Ia(1 -30)inhibin to JLB #492 antiserum by serial dilutions of cow serum and follicular fluid were parallel to the inhibin-a standard curve. Briefly, the tubes were incubated at room temperature for 3 days with 50 pl of serum or follicular fluid diluted wtth 20 pl of assay buffer (PBS containing 0.1% bovine serum albumin, pH 7.4) and antiserum (100 t~l of 1: 40 000 final dilution of JLB #492) added on Day 1. Labeled hormone ( 25,000 cpm/lOO pl) was added on Day 2. The second antibody (sheep anti-rabbi gamma globulin; 1200 in 10% PEG-PBS) was added on Day 3, incubated for 1 hour at room temperature, and then centriiged at 3500 x g for 30 minutes to separate free from antibody-bound hormone. The intra- and inter-assay CVs were 10 and 15.2%, respectively. The assay sensitivity was 0.2 ng/ml. Statistical Analysis Mean inhibin, FSH and LH concentrations, pulse frequencies and amplitudes between treatments were analyzed by ANOVA for repeated measurements wlth time as a subplot (i.e., blood sampling period; 24). A pulse was defined by the following criteria: 1) the hormone increase was greater than 50% of the previous nadir (21), 2) the hormone level remained elevated for two sampling periods (25), and 3) the peak was followed by two declining points or one declining point of at least 30% of the pulse amplitude (21). Amplitude was defined as the diierence between the maximal value reached during a pulse and the nadir preceding the pulse (12). Pearson’s coefficient of correlations were made between follicular fluid inhibin and gonadotropin concentrations, serum inhibin and gonadotropin concentrations, follicular fluid inhibin and serum inhibin concentrations, and follicular fluid gonadotropin and serum gonadotropin concentrations (25). Chi-square analysis was used to determinedierertce between the proportionof cows that exhibiied pulses of inhibin,FSH or LH in both treated groups (26).
Theriogenology
812 RESULTS
There was a tendency for mean concentrations of serum inhibin to be higher and FSH to be lower in CCOD cows than in cyclic (control) cows (P c 0.1; Figure 1 a and b); whereas, mean concentrations of serum LH were similar (P > 0.05; Figure lc). There were no correlations (P > 0.06) between: 1) serum gonadotropin and inhibin concentrations, 2) follicular fluid gonadotropin and serum gonadotropin, or 3) follicular fluid inhibin and serum inhlbin concentrations. In contrast, follicular fluid concentrations of FSH and LH were negatively correlated to follicular fluid inhibin concentration (r = -0.79 and r = -0.66, respectively; P -z 0.05). Concentrations of inhibin were increased and FSH were decreased in the follicular fluid of CCOD cows compared with that of the control cows (P < 0.06; Table 1); however, follicular fluid LH concentrations were not diierent (P > 0.05; Table 1). For inhibin and LH, neither pulse frequencies or pulse amplftudes differed between CCOD and control cows (P > 0.05; Table 2). Similarly, there was no difference in FSH pulse amplitude between these 2 groups (Table 2). In There also was a contrast, FSH pulse frequency was lower (P < 0.06; Table 2). tendency for fewer CCOD cows to exhibit FSH pulses and a higher number of CCOD cows to exhibit lnhibin pulses than control cows (P < 0.1; Table 2). DISCUSSION The present study demonstrates that follicular fluid of cystic follicles from CCOD cows contains greater concentrations of inhibin than preovulatory follicles of control cyclic cows. In agreement with these data, higher inhibin concentrations in follicular fluid of cystic follicles have also been reported in women (17,19,20) and rats (20). lt is well known that granulosa cells are the major source of inhibin (15). A positive correlation between inhibin concentrations in follicular fluid and the number of granulosa cells per follicle has been reported in cyclic cows (27). Similarly, a reduced number of granulosa calls has been associated with lowered in vitro production of inhibin and estradlol-178 in bovine atretlc follicles than in healthy follicles (27). Even though there are fewer granulosa cells in cystic follicles of women and cows compared to normal non-atretlc follicles (10, 26) it has been suggested that there is a hyperfunction of granulosa cell activity in cystic follicles, resuiting in a higher production of inhibin per cell (16). A previous study has shown the presence of large amounts of monomeric inhibin a subunit, which is biologically inactive in bovine follicular fluid (16). In this study, the radioimmunoassay for inhibin measurements used an antisera raised against porcine a subunft and therefore would bind the bioactive inhibin dimeric u and 6 subunits as well as the inactive inhibin monomeric a subunit. Therefore, the results obtained in this inhibin RlA reflects the immunoreactivity but not necessarily the biological activity of inhibin present In the follicular fluid. In contrast to inhibin, concentrations of FSH in follicular fluid of cystic follicles were reduced compared to the preovulatory follicles of cycling cows. lt has previously been reported that the FSH concentration within follicles of cycling sheep was correlated with the functional status of the follicles, as determined by the amounts and ratios of follicular fluid androgens and estrogens (29). Specifically, the highest concentrations of FSH were present in follicles with high concentrations of estrogen (estradiol: androstenedione ratio > 1); whereas the lowest FSH concentrations were found in follicles wlth low concentrations of estrogen (estradiol: androstenedlone ratio < 1) (29). In the present study, follicular fluid concentrations of FSH were negatively correlated to
813
Theriogenology
b)
60
1
0
0
60
120 TIME
180
240
CMINUTESI
Figure 1. Serum concentrations of a) inhibin, b) FSH and c) LH in CCOD cows (n=15) and cyclic cows (control; n=6). Samples were collected every 15 minutes for 4 hours. Results are presented as mean rt SEM.
814
Table 1.
Theriogenology Follicular fluid concentrations of inhibin, FSH and LH from cystic follicles of CCOD cows (n=15) and preovulatory follicles of cyclic cows (control; n=6)
Follicular fluid concentration Chronic cystic ovarian diseased cows (CCOD)
Control cows
Hormone
Inhibin (pg/ml) FSH (ng/ml) LH (ng/ml)
4.2 75.0 0.6
4.9 k 0.1 * 22.6 + 4.0 *** 0.5 zk 0.1
-t 0.3 -t 7.0 zk 0.1
Mean 2 SEM * P c 0.05 *** P < 0.001
Table 2.
Mean concentrations, frequency (number of pulses/4 hours) and pulse amplitude of serum inhibin, FSH and LH in CCOD cows (n=15) and in cyclic cows (control; n=6). Number in parentheses represents the number of cows in each group which exhibited pulses.
Hormone
Control cows
Chronic cystic ovarian diseased cows (CCOD)
LH: mean (ng/ml) frequency (pulses/4 hours) pulse amplitude (ng/ml)
1.1 k 0.3 1.6 f 0.5 1.5 * 0.7
(5/6)
0.6 k 0.2 2.0 k 0.4 (13/15) 1.5 + 0.6
FSH: mean (rig/ml) frequency (pulses/4 hours) pulse amplitude (ng/ml)
44.9 2 2.3 0.6 k 0.2 34.7 k 7.6
(3/5)
36.6 + 3.6 0.2 + 0.2 * (3/13) 36.3 +- 7.5
INHIBIN: mean (ng/ml) frequency (pulses/4 hours) pulse amplitude (ng/ml)
1.2 -c 0.5 0.3 + 0.3 2.2 f 1.1
(l/6)
1.6 k 0.3 0.7 f 0.2 (6/15) 2.1 f 0.4
Mean -t SEM P < 0.05
l
Theriogenology
815
inhibin concentrations. It has previously been shown that inhibin can suppr0ss FSH-induced granulosa cell estrogen production (15). Therefore, the higher production of inhibin in the follicles of CCOD cows may result in a greater Suppression Of FSH-induced estrogen production and a shift in follicular steroidogenesis to the production of androgens, as previously observed in CCOD cows (12). There was a tendency for serum concentrations of inhibin to be higher and FSH to be lower in CCOD cows than in cyclic cows (P < 0.1). A decrease in FSH pulse frequency (P c 0.05) in CCOD cows was also observed, which probably explains the tendency for lowered mean serum FSH concentration. lt was observed that the mean FSH values of the CCOD and control cows were greater than the FSH pulse amplitude values. This could be explained by the fact that not all the cows showed pulses and those that did had a lower basal level of FSH than the non-pulsing cows. In fact, only 23% of the CCOD cows exhibited FSH pulses (during the ehour sampling period) compared to 60% of the cyclic cows. Although there was no difference in inhibin pulse frequency between CCOD and cyclic cows, 65% more of the CCOD cows exhibiied inhibin pulses than cyclic cows. However, in cyclic sheep, episodic secretion of inhibin has been detected in the ovarian vein, but not in the peripheral circulation (36). Although it has previously been reported that there is no difference in mean serum concentration of FSH in CCOD cows compared with cyclic cows (12), the number of CCOD cows in that study was smaller than in this present study. No difference in serum FSH was observed in polycystfc ovarian diseased women compared to normal women (31). However, in polycystic ovarian diseased rats, high levels of inhibin in follfcular fluid from cystic follicles was found in association with a decreased concentration of serum FSH (26). Likewise, passive immunization of polycystic ovarian diseased rats with inhibin antiserum increased serum FSH and induced follicular maturation, ovulation, and the reestablishment of normal cyclic&y (26). Follicle stimulating hormone is known to be important for stimulating granulosa cell LH and FSH receptor populations and estrogen production (32). In CCOD cows, concentrations of gonadotropin receptors and estradiol secretion were both reduced compared to that of cyclic cows (12). Therefore, based on the data of Brown et al. (12) and our present study, we propose a model to explain how the CCOD condition is maintained in dairy cows. First, an increase in inhibin production from the cystic follicles inhibits the pituitary release of FSH. Then a reduction in FSH at the ovarian level reduces the numbers of follicular LH and FSH receptors and resutts in abnormal follicular steroidogenesis (estradiol: androstenedione ratio < l), which are the conditions characteristic of the cystic follicles in CCOD cows. Finally, the lack of adequate numbers of follicular gonadotropin receptors prevents the LH induction of ovulation and reestablishment of normal ovarian cyclicity. Since it has previously been shown that treatment of CCOD cows with exogenous FSH can induce normal follicular development (12) it appears that inadequate FSH secretion may indeed be a causative factor in the CCOD syndrome. While this model attempts to explain the hormonal mechanisms that maintain the CCOD syndrome in dairy cows, it cannot explain what initially causes the cyst formation. Since these observations are based on a condition that had been present for 10 months or longer, the endocrinological patterns present during cyst formation may no longer be present. Thus, future studies need to be conducted to verify whether this model is also applicable during cyst formation.
816
Theriogenology
REFERENCES 1. Kesler, D.J. and Garverick, H.A. Ovarian cysts in dairy cattle: a review. J. Anim. Sci. &1147-1159 (1962). 2.
Eyestone, W.H. and Ax, R.L. A review of ovarian follicular cysts in cows, with comparisons to the condition in women, rats and rabbis. Theriogenology p:169125 (1964).
3. Casida, L.E. and Chapman, A.B. Factors affecting the incidence of cystic ovaries in a herd of Holstein cows. J. Dairy Sci. &1266-1265 (1951). 4. Bierschwal, C.J., Garverick, H.A., Martin, C.E., Youngquist, R.S., Cantley, T.C. and Brown, M.D. Clinical response of dairy cows with ovarian cysts to GnRH. J. Anim. Sci. 41:1660-l 665 (1975). 5.
Elmore, R.G., Bierschwal, C.G., Youngquist, R.S., Cantley, T.C., Kesler, D.J. and Garverick, H.A. Clinical responses of dairy cows with ovarian cysts following treatment with 10,669 IU hCG or 166 mcg GnRH. Vet, Med. Small Anim. Clin. a:1346-1349 (1975).
6. Seguin, B.E., Convey, E.M. and Oxender, W.D. Effect of GnRH and hCG in cows with ovarian folllcular cysts. Amer. J. Vet. Res. 37:153-l 57 (1976). 7. Garverick, H.A., Kesler, D.J., Cantley, T.C., Elmore, B.G., Youngquist, R.S. and Bierschwal, C.J. Hormone response of dairy cows with ovarian cysts after treatment with hCG or GnRH. Theriogenology 6:413426 (1976). 6.
Kesler, D.J., Garverick, H.A., Elmore, R.G., Youngquist, R.S. and Bierschwal, C.J. Reproductive hormones associated with the ovarian cyst response to GnRH. Theriogenology a:1 69-l 14 (1979).
9.
Tanabe, T.V. and Brofee, R.D. Treatment of cystic ovarian follicles in dairy cows with chorionic gonadotropin. Theriogenology j&497-51 2 (1962).
10.
Brown, EM., Elmore, R.G., Garverick, H.A. and Kesler, D.J. Gonadotropin releasing hormone treatment of dairy cows with ovarian cysts. II. Histology of ovarian cyst walls. Theriogenology 17: 669696 (1962).
11.
DeSilva, M. and Reeves, J.J. Hypothalamic-pituitary function in chronically cystic and regularly cycling dairy cows. Biol. Reprod. 3&?64269 (1966).
12.
Brown, J.L., Schoenemann, H.M. and Reeves, J.J. Effect of FSH treatment on LH and FSH receptors in chronic cystic-ovarian diseased dairy cows. J. Anim. Sci. 63:1663-1071 (1966).
13.
Short R.V. Steroid concentrations in normal follicular fluid and ovarian cyst fluid from cows. J. Reprod. Fertil. A:2745 (1962).
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14. Erickson, G.G. and Hsueh, A.J.W. Secretion of inhibin by rat granulosa cells in vitro. Endocrinology 103:1988-l 983 (1978). 15. Ylng, S.Y. Inhibins, activins and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocrinol. Rev. 9:287-293 (1988). 18. Knight, P.G., Beard. A.J., Wrathall, J.H.M., Castillo, R.J. Evidence that the bovine ovary secretes large amounts of monomeric inhibin subunit 8 and its isolation from bovine follicular fluid. J. Mol. Endocrinol. 2:189-208 (1988). 17. Daume, E., Chari, S., Sturm, G., Hillensjo, T., Mangnusson, C. Non-steroidal ovarian inhibitors in human follicular fluid. In: Rolland, R., Van Hall, E.V., Hillier, S.G., McNatty, KP. and Schoemaker, J. (eds), Follicular Maturation and Ovulation. Excerpta Medica, Amsterdam, 1982, ~~284-298. 18. Tanabe, K., Gagliano, P., Channing, C.P., Nakamura, Y., Yoshimura, Y., lizuka, R., Fortuny, A., Sulewski, J. and Rezai, N. Levels of inhibin-F activity and steroids in human follicular fluid from normal women and women with polycystic ovarian disease. J. Clin. Endocrinol. Metab. 57324-31 (1983). 19. Chari, S., Daume, E., Strum, G., Vanupel, H. and Schuler, I. Regulators of steroid secretion and inhibin activity in human ovarian follicular fluid. Mol. Cell. Endocrinol. 41:137-l 45 (1985). 28. Hurkadfi, K. and Sheth, A.R. Elevation of FSH levels by antibodies to inhibin and its potential use in reproductive pathophysiology. In: Sheth, A.R. (ed), Inhibins: Isolation, Estimation and Physiology, Vol. II. CRC Press, Boca Raton, FL, 1987, pp. 85-79. 21. Acosta, B., Tarnavsky, G.K, Platt, T.E., Hamernick, D.L., Brown, J.L., Schoenomann, H.M. and Reeves, J.J. Nursing enhances the negative effect of estrogen on LH and FSH release in the cow. J. Anim. Sci. =:15381538 (1983). 22. Golter, T.D., Reeves, J.J., O’Mary, C.C., Arimura, A. and Schally, A.V. Serum LH levels in bulls treated with synthetic luteinizing hormone-releasing hormone/ follicle stimulating hormone-releasing hormone (LH-RH/FSH-RH). J. Anim. Sci. x:123127(1973). 23. Brown, J.L. and Chakraborty, P.K. Comparison of compensatory pituitary and testicular responses to hemicastration between prepubertal and mature rats. J. Androl. 12:119-125 (1991). 24. Gill, J.L. and Hafs, H.D. Analysis of repeated measurements of animals. J. Anim. Sci. 33331-338 (1971). 25. Steel, R.G. and Torrie, J.H. Linear correlation. In: Napier, C., Maisel, J.W. (eds), Principles and Procedures of Statistics. McGraw-Hill Co., San Francisco, 1980, pp.272-284.
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26. Goodman, R.L.and Karsh, F.J. Pulsatile secretion of luteinizing hormone: differential suppression by ovarian steroids. Endocrinology =:1286-1290 (1980). 27. Henderson, K.M., Franchimont, P., Charlet-Renard, Ch. and McNatty, K.P. Effect of follicular atresia on inhibin production by bovine granulosa cells in vitro and inhibin concentrations in the follicular fluid. J. Reprod. Fertil. 2:1-8 (1984). 26. Al-Dahash, S.Y.A. and David, J.S.E. Anatomical features of cystic ovaries in cattle found during an abbatoir survey. Vet. Rec. m:O-4 (1977). 29. McNatty, K.P., Gibb, M., Dobson, C., Thurley,. DC. and Findlay, J.K. Changes in the concentration of gonadotrophic and steroidal hormones in the antral fluid of ovarian follicles throughout the oestrous cycle of the sheep. Aust. J. Biol. Sci. %:67-80 (1981). 30. McNeilly, A.S. and Baird, D.T. Episodic secretion of inhibin into the ovarian vein during the follicular phase of the oestrous cycle in the ewe. J. Endocrinol. =:287292 (1989). 31. Waldstreicher, J., Santoro, N.F., Hall, J.E., Filicori, M. and Crowley, W.F. Hyperfunction of the hypothalamic-pituitary axis in women with polycystic ovarian J. Clin. disease: indirect evidence for partial gonadotroph desensitization. Endocrinol. Metab. B:165-172 (1988). 32. Hsueh, A.J.W., Adashi, E.Y., Jones, P.B.C. and Welsh, T.H. Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocrinol. Rev. 5:76-i 27 (1984).
40:809-818,1993
ELEVATED INHIBIN CONCENTRATION IN THE FOLLlCUlAFl FLUID OF DAIRY COWS WlTH CHRONIC CYSTIC OVAMAN DISEASE
S. Roberge,la J.L. Brown* and J.J. Reeves’
’ Department of Animal Science, Washington State University Pullman WA 991646332 *Conservation and Research Center, Front Royal VA 22636
Received for publication: hxmnber 15, 2992 Accepted: June 8, 199.3 ABSTRACT This study compared serum and follicular fluid inhibin and gonadotropin profiles between chronic cystic ovarian diseased (CCOD) and normal cyclic dairy cows. Blood samples and follicular fluid were collected from CCOD cows (n=15) and cyclic cows in the follicular phase of the estrous cycle (control, n=6) and analyzed for inhibin, follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations. There was a significant increase in inhibin and a decrease in FSH and LH concentrations in the follicular fluid of CCOD cows compared with those of cyclic cows (P e 0.05). Mean serum inhibin, FSH and LH concentrations between CCOD and cyclic cows were not different (P > 0.05) however, there was a tendency for serum inhibin to be higher and FSH to be lower in CCOD cows compared to cyclic animals (P e 0.1). The FSH pulse frequency also was lower in CCOD cows than in cyclic cows (P < 0.05). These data suggest that increased production of inhibin from cystic follicles of CCOD cows alters pituitary FSH secretion and subsequently reduces the concentration of FSH in follicular fluid. As a result, decreased FSH stimulation at the ovarian level could ultimately lead to the reduction in follicular LH and FSH receptor concentrations, resuiting in abnormal follicular steroidogenesis in CCOD dairy cows.
Key words: inhibin, FSH, cyst, follicular fluid, cystic cow Acknowledgments Scientific paper no. 6656. College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA 99164. ‘Present address and correspondence: Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, CANADA Ni G 2Wl. FAX: (519) 767-6573.
Copyright Q 1993 Butterworth-Heinemann
Theriogenology
810 INTRODUCTION
Ovarian cysts in dairy cows are generally defined as anovulatory follicular structures, at least 2.5 cm in diameter, that persist for 10 days or more in the absence of a corpus luteum (CL; 1,2). Approximately 80% of cows with cystic ovarian disease (COD) respond to treatment with human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRH; 3-9). These treatments induce the release of endogenous luteinizing hormone (LH) and cause luteinization of the cystic structure and a reestablishment of ovarian cyclicity within 21 to 24 days of treatment (4-8). However, 29% of COD cows do not respond to hormone treatment and are referred to as chronic cystic-ovarian diseased (CCOD) cows (5,8,8). Some studies have reported that the cystic condition is apparently not related to a pituitary dysfunction, since neither serum LH or FSH secretion, nor pituitary LHRH receptor concentrations are altered in cystic cows compared with that of cyclic cows (8, 9-l 1). Instead, it has been suggested that the inability of CCOD cows to respond to hormonal treatment is due to a specific dysfunction at the ovarian level (12). In that study, a significant decrease in both LH and FSH receptors in the ovarian wall and follicular granulosa cells was observed in CCOD cows compared with that of cyclic cows. There was also a higher ratio of androgens to estrogens in the fluid of cystic follicles compared to that of non atretric follicles of cyclic cows (12). Reduced follicular aromatase activity in cystic follicles, resulting in reduced estrogen production, has also been reported (2,13-l 4). In addition to producing steroids, granulosa cells also produce a glycoprotein hormone, inhibin, which plays an important role in suppressing pituitary FSH secretion both in vivo and in vitro (15). lnhibin also directly inhibits FSH-induced granulosa cell estrogen production (15). It has been demonstrated that there is presence of large amounts of inhibin in bovine follicular fluid (18). In polycystic ovarian diseased women, higher concentrations of inhibin in follicular fluid of cystic follicles have been observed than in normal follicles (17-l 9). Likewise, high concentrations of inhibin in follicular fluid from rat cystic follicles were accompanied by a decrease in serum FSH secretion (29). There is, as yet, no report on the role of inhibin in CCOD dairy cows. Therefore, the objectives of this research project were to compare serum and follicular fluid inhibin, FSH and LH profiles between CCOD and normal cyclic cows. MATERIALS AND METHODS Animals Fifteen lactating Holstein cows, diagnosed as cystic by veterinarians using palpation per rectum, were used in this study. These were termed CCOD cows because they had been treated an average of 8 times with hCG or LHRH without response and had been in a chronic cystic condition for an average of 10 months. All cows were lactating and were milked twice daily until termination of the study. Six noncystic, cyclic Holstein control cows in the luteal phase of the estrous cycle were injected with 35 to 50 mg of PGFsaband were slaughtered approximately 48 to 50 hours after injection, to ensure that they were in the follicular phase at the time of slaughter. Samples from 7 CCOD cows and from all control cows were utilized in a previous study (12). Samples from an additional 8 CCOD cows were collected within the same year of the above study. b Lutalyse, Upjohn Co., Kalamazoo,
MI.
Theriogenolog
y
Blood Sampling and Follicular Fluid Collection All cows were fitted with jugular catheters the day before slaughter and blood samples were collected every 15 minutes for 4 hours. Approximately 12 hours after collection, all the animals were slaughtered, and the ovaries were removed, placed on ice, and examined for follicular and luteal development. Ovaries from the CCOD cows contained singular cysts that exceeded 2.5 cm in diameter, and had no functional CL present. Control cows were considered to be follicular if the ovary contained a follicle 1.5 to 2.0 cm in diameter. Follicular fluid from control and cystic follicles was aspirated using a syringe fitted with a 2O-gauge needle. Serum and follicular fluid were stored at -20°C until assayed for inhibin, FSH and LH concentrations. Radioimmunoassays Serum and follicular fluid concentrations of FSH were determined by a double antibody radioimmunoassay (FM) as described previously using USDA-bFSH-B-1 asthe hormone standard (21). Intra- and inter-assay coefficients of variation (CV) were 8.6 and 11.2%, respectively. Assay sensitivity was 7.5 ng/ml. Concentrations of LH were determined in a single MA as described previously using NIH-LH-B7 as the hormone standard (22). Intra-assay CV was 5%. The sensitivity of the assay was 0.3 ng/ml. Concentrations of inhibin were determined by a RlA previously described (23), using antiserum raised against a synthetic porcine a(l-30) inhibin fragment (JLB #492) and the same synthetic a(1-30) fragment as the labeled hormone. Displacement of ‘“Ia(1 -30)inhibin to JLB #492 antiserum by serial dilutions of cow serum and follicular fluid were parallel to the inhibin-a standard curve. Briefly, the tubes were incubated at room temperature for 3 days with 50 pl of serum or follicular fluid diluted wtth 20 pl of assay buffer (PBS containing 0.1% bovine serum albumin, pH 7.4) and antiserum (100 t~l of 1: 40 000 final dilution of JLB #492) added on Day 1. Labeled hormone ( 25,000 cpm/lOO pl) was added on Day 2. The second antibody (sheep anti-rabbi gamma globulin; 1200 in 10% PEG-PBS) was added on Day 3, incubated for 1 hour at room temperature, and then centriiged at 3500 x g for 30 minutes to separate free from antibody-bound hormone. The intra- and inter-assay CVs were 10 and 15.2%, respectively. The assay sensitivity was 0.2 ng/ml. Statistical Analysis Mean inhibin, FSH and LH concentrations, pulse frequencies and amplitudes between treatments were analyzed by ANOVA for repeated measurements wlth time as a subplot (i.e., blood sampling period; 24). A pulse was defined by the following criteria: 1) the hormone increase was greater than 50% of the previous nadir (21), 2) the hormone level remained elevated for two sampling periods (25), and 3) the peak was followed by two declining points or one declining point of at least 30% of the pulse amplitude (21). Amplitude was defined as the diierence between the maximal value reached during a pulse and the nadir preceding the pulse (12). Pearson’s coefficient of correlations were made between follicular fluid inhibin and gonadotropin concentrations, serum inhibin and gonadotropin concentrations, follicular fluid inhibin and serum inhibin concentrations, and follicular fluid gonadotropin and serum gonadotropin concentrations (25). Chi-square analysis was used to determinedierertce between the proportionof cows that exhibiied pulses of inhibin,FSH or LH in both treated groups (26).
Theriogenology
812 RESULTS
There was a tendency for mean concentrations of serum inhibin to be higher and FSH to be lower in CCOD cows than in cyclic (control) cows (P c 0.1; Figure 1 a and b); whereas, mean concentrations of serum LH were similar (P > 0.05; Figure lc). There were no correlations (P > 0.06) between: 1) serum gonadotropin and inhibin concentrations, 2) follicular fluid gonadotropin and serum gonadotropin, or 3) follicular fluid inhibin and serum inhlbin concentrations. In contrast, follicular fluid concentrations of FSH and LH were negatively correlated to follicular fluid inhibin concentration (r = -0.79 and r = -0.66, respectively; P -z 0.05). Concentrations of inhibin were increased and FSH were decreased in the follicular fluid of CCOD cows compared with that of the control cows (P < 0.06; Table 1); however, follicular fluid LH concentrations were not diierent (P > 0.05; Table 1). For inhibin and LH, neither pulse frequencies or pulse amplftudes differed between CCOD and control cows (P > 0.05; Table 2). Similarly, there was no difference in FSH pulse amplitude between these 2 groups (Table 2). In There also was a contrast, FSH pulse frequency was lower (P < 0.06; Table 2). tendency for fewer CCOD cows to exhibit FSH pulses and a higher number of CCOD cows to exhibit lnhibin pulses than control cows (P < 0.1; Table 2). DISCUSSION The present study demonstrates that follicular fluid of cystic follicles from CCOD cows contains greater concentrations of inhibin than preovulatory follicles of control cyclic cows. In agreement with these data, higher inhibin concentrations in follicular fluid of cystic follicles have also been reported in women (17,19,20) and rats (20). lt is well known that granulosa cells are the major source of inhibin (15). A positive correlation between inhibin concentrations in follicular fluid and the number of granulosa cells per follicle has been reported in cyclic cows (27). Similarly, a reduced number of granulosa calls has been associated with lowered in vitro production of inhibin and estradlol-178 in bovine atretlc follicles than in healthy follicles (27). Even though there are fewer granulosa cells in cystic follicles of women and cows compared to normal non-atretlc follicles (10, 26) it has been suggested that there is a hyperfunction of granulosa cell activity in cystic follicles, resuiting in a higher production of inhibin per cell (16). A previous study has shown the presence of large amounts of monomeric inhibin a subunit, which is biologically inactive in bovine follicular fluid (16). In this study, the radioimmunoassay for inhibin measurements used an antisera raised against porcine a subunft and therefore would bind the bioactive inhibin dimeric u and 6 subunits as well as the inactive inhibin monomeric a subunit. Therefore, the results obtained in this inhibin RlA reflects the immunoreactivity but not necessarily the biological activity of inhibin present In the follicular fluid. In contrast to inhibin, concentrations of FSH in follicular fluid of cystic follicles were reduced compared to the preovulatory follicles of cycling cows. lt has previously been reported that the FSH concentration within follicles of cycling sheep was correlated with the functional status of the follicles, as determined by the amounts and ratios of follicular fluid androgens and estrogens (29). Specifically, the highest concentrations of FSH were present in follicles with high concentrations of estrogen (estradiol: androstenedione ratio > 1); whereas the lowest FSH concentrations were found in follicles wlth low concentrations of estrogen (estradiol: androstenedlone ratio < 1) (29). In the present study, follicular fluid concentrations of FSH were negatively correlated to
813
Theriogenology
b)
60
1
0
0
60
120 TIME
180
240
CMINUTESI
Figure 1. Serum concentrations of a) inhibin, b) FSH and c) LH in CCOD cows (n=15) and cyclic cows (control; n=6). Samples were collected every 15 minutes for 4 hours. Results are presented as mean rt SEM.
814
Table 1.
Theriogenology Follicular fluid concentrations of inhibin, FSH and LH from cystic follicles of CCOD cows (n=15) and preovulatory follicles of cyclic cows (control; n=6)
Follicular fluid concentration Chronic cystic ovarian diseased cows (CCOD)
Control cows
Hormone
Inhibin (pg/ml) FSH (ng/ml) LH (ng/ml)
4.2 75.0 0.6
4.9 k 0.1 * 22.6 + 4.0 *** 0.5 zk 0.1
-t 0.3 -t 7.0 zk 0.1
Mean 2 SEM * P c 0.05 *** P < 0.001
Table 2.
Mean concentrations, frequency (number of pulses/4 hours) and pulse amplitude of serum inhibin, FSH and LH in CCOD cows (n=15) and in cyclic cows (control; n=6). Number in parentheses represents the number of cows in each group which exhibited pulses.
Hormone
Control cows
Chronic cystic ovarian diseased cows (CCOD)
LH: mean (ng/ml) frequency (pulses/4 hours) pulse amplitude (ng/ml)
1.1 k 0.3 1.6 f 0.5 1.5 * 0.7
(5/6)
0.6 k 0.2 2.0 k 0.4 (13/15) 1.5 + 0.6
FSH: mean (rig/ml) frequency (pulses/4 hours) pulse amplitude (ng/ml)
44.9 2 2.3 0.6 k 0.2 34.7 k 7.6
(3/5)
36.6 + 3.6 0.2 + 0.2 * (3/13) 36.3 +- 7.5
INHIBIN: mean (ng/ml) frequency (pulses/4 hours) pulse amplitude (ng/ml)
1.2 -c 0.5 0.3 + 0.3 2.2 f 1.1
(l/6)
1.6 k 0.3 0.7 f 0.2 (6/15) 2.1 f 0.4
Mean -t SEM P < 0.05
l
Theriogenology
815
inhibin concentrations. It has previously been shown that inhibin can suppr0ss FSH-induced granulosa cell estrogen production (15). Therefore, the higher production of inhibin in the follicles of CCOD cows may result in a greater Suppression Of FSH-induced estrogen production and a shift in follicular steroidogenesis to the production of androgens, as previously observed in CCOD cows (12). There was a tendency for serum concentrations of inhibin to be higher and FSH to be lower in CCOD cows than in cyclic cows (P < 0.1). A decrease in FSH pulse frequency (P c 0.05) in CCOD cows was also observed, which probably explains the tendency for lowered mean serum FSH concentration. lt was observed that the mean FSH values of the CCOD and control cows were greater than the FSH pulse amplitude values. This could be explained by the fact that not all the cows showed pulses and those that did had a lower basal level of FSH than the non-pulsing cows. In fact, only 23% of the CCOD cows exhibited FSH pulses (during the ehour sampling period) compared to 60% of the cyclic cows. Although there was no difference in inhibin pulse frequency between CCOD and cyclic cows, 65% more of the CCOD cows exhibiied inhibin pulses than cyclic cows. However, in cyclic sheep, episodic secretion of inhibin has been detected in the ovarian vein, but not in the peripheral circulation (36). Although it has previously been reported that there is no difference in mean serum concentration of FSH in CCOD cows compared with cyclic cows (12), the number of CCOD cows in that study was smaller than in this present study. No difference in serum FSH was observed in polycystfc ovarian diseased women compared to normal women (31). However, in polycystic ovarian diseased rats, high levels of inhibin in follfcular fluid from cystic follicles was found in association with a decreased concentration of serum FSH (26). Likewise, passive immunization of polycystic ovarian diseased rats with inhibin antiserum increased serum FSH and induced follicular maturation, ovulation, and the reestablishment of normal cyclic&y (26). Follicle stimulating hormone is known to be important for stimulating granulosa cell LH and FSH receptor populations and estrogen production (32). In CCOD cows, concentrations of gonadotropin receptors and estradiol secretion were both reduced compared to that of cyclic cows (12). Therefore, based on the data of Brown et al. (12) and our present study, we propose a model to explain how the CCOD condition is maintained in dairy cows. First, an increase in inhibin production from the cystic follicles inhibits the pituitary release of FSH. Then a reduction in FSH at the ovarian level reduces the numbers of follicular LH and FSH receptors and resutts in abnormal follicular steroidogenesis (estradiol: androstenedione ratio < l), which are the conditions characteristic of the cystic follicles in CCOD cows. Finally, the lack of adequate numbers of follicular gonadotropin receptors prevents the LH induction of ovulation and reestablishment of normal ovarian cyclicity. Since it has previously been shown that treatment of CCOD cows with exogenous FSH can induce normal follicular development (12) it appears that inadequate FSH secretion may indeed be a causative factor in the CCOD syndrome. While this model attempts to explain the hormonal mechanisms that maintain the CCOD syndrome in dairy cows, it cannot explain what initially causes the cyst formation. Since these observations are based on a condition that had been present for 10 months or longer, the endocrinological patterns present during cyst formation may no longer be present. Thus, future studies need to be conducted to verify whether this model is also applicable during cyst formation.
816
Theriogenology
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12.
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13.
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