- Email: [email protected]
Circulating concentrations of immunoreactive inhibin and FSH during the estrous cycle of mares
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CIRCULATINGCONCENTRATIONSOF IMMUNOREACTIVE INHIBINAND FSH DURINGTHE ESTROUSCYCLEOF MARES D. R. Bergfelt, MS;1 B. G. Mann, BS; 2 N. B. Schwartz, PhD; 2 and O. J. Ginther, VMD, PhD 1
SUMMARY
A heterologous radioimmunoassay developed to measure inhibin in rat plasma was validated and used to characterize changes in peripheral concentrations of immunoreactive inhibin (ir-inhibin) in relation to follicle stimulating hormone (FSH) concentrations during the estrous cycle of mares. The primary antiserum used in the assay was developed against a synthetic porcine inhibin ct-subunit [(1-26)-Gly-Tyr] fragment. The same synthetic peptide was used for preparation of standards and tracer. Slopes of the dose-response curves for pooled estrus and diestrus mare plasma and equine follicular fluid were similar to the slopes for the porcine inhibin t~subunit standard curve and porcine follicular fluid doseresponse curve. Twelve mares were bled once daily beginning when diameter of the largest follicle reached >_25 mm and continuing until 3 days after the end of an interovulatory interval (ovulation=Day 0). Each of the 12 interovulatory intervals were normalized to the mean length of the interovulatory interval (22.2 days; range, 19 to 26). There was an effect Authors' addresses: 1 Department of Veterinary Science, University of Wisconsin-Madison, Madison, Wisconsin 53706; 2Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208. Send reprint requests to Dr. Ginther. Acknowledgments: Supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and Equiculture, Inc., Cross Plains, Wisconsin. The authors thank L. Kulick for preparation of figures, M. Westphal for manuscript preparation, and Drs, C. Rivier and W. Vale for the antigen and antiserum used in the inhibin assay.
Volume 11, Number 6, 1991
of day for concentrations of ir-inhibin (P<0.001) and FSH (P<0.006). Significant mean changes were as follows: 1) irinhibin decreased between Days 0 and 1, whereas FSH increased between Days 0 and 5; and 2) ir-inhibin increased between Days 7 and 12, whereas FSH decreased between Days 11 and 14. Mean concentrations of ir-inhibin and FSH were negatively correlated (r=--0.548; P<0.002). In conclusion, mean peripheral concentrations of ir-inhibin and FSH were inversely related during the estrous cycle of mares.
INTRODUCTION
Follicular fluid of domestic species (porcine, bovine, ovine) contains a component termed inhibin that has the capacity to preferentially suppress follicle stimulating hormone (FSH).4,5 Inhibin has been defined as a glycoprotein hormone consisting of an t~-subunit and 1 of 2 distinct lsubunits, 8k or I~. 2The complete amino acid sequence of the precursor of porcine, bovine, rat, and human inhibin has been predicted from complementary DNA sequences, resulting in the development of sensitive radioimmunoassays.4'5 An antiserum directed against a syntheticporcine inhibin t~-subunit fragment has been used to measure levels of inhibin in rat plasma.l.20 Circulating concentrations of inhibin have been characterized during the estrous cycle in a limited number of sows,12 cows,6.12.18and ewes.7 Circulating concentrations of inhibin in the mare have not been reported. However, immu-
319
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.
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=E
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Figure 1. Radioimmunoassay dose-response curves for pooled estrus and diestrus mare plasma and equine and porcine follicular fluid in relation to the porcine inhibin c¢subunit [(1-26)-Gly-Tyr] standard curve. The slopes of the equine plasma and follicular fluid, between 20 and 90% bound, were not different (P>0.05) from the slopes of the porcine inhibin c~-subunit standard curve and the porcine follicular fluid dose-response curve. noblot results from tumor-conditioned media indicated that inhibin c¢-and I]A-subunitsare produced by equine granulosatheca cell tumors.1z Aproteinaceous FSH-inhibiting component in equine follicularfluid has been reported,3,14but it is not known if it is similar to the inhibin component of porcine, bovine, and ovine follicular fluid. The objectives of this study were to validate an inhibin assay for mares using a heterologous radioimmunoassay developed for rat plasma1,2° and to characterize changes in peripheral concentrations of immunoreactive inhibin (ir-inhibin) in relation to FSH concentrations during the eslxous cycle of mares.
MATERIALS
AND METHODS
Twelve riding-type horse mares 4 to 16 years of age and weighing 400 to 550 kg were used during May to August. Breeds were not known, but appeared to be primarily Quarter Horse and Appaloosa based on size, conformation, and color pattems. Transrectal ultrasonic examination of the ovaries was done as described10 to determine the day of ovulation at 320
,
.
,
,
~
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.
,
.
,
.
,
,
,
.
,
.
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.
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.
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.
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.
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.
o
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.
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Figure 2. Mean (_+sem;n=l 2) circulating concentrations of irinhibin and FSH during the interovulatory interval in mares. Ovulation (OV) is Day O. There was an effect of day for both ir-inhibin (P
each end of an interovulatory interval (interval between the ovulations of successive ovulatory periods). If double asynchronous ovulations occurred during a given ovulatory period, the first ovulation was used to assign a beginning and end to the interovulatory interval. Mares were examined and bled once daily beginning when diameter of the largest follicle reached >_25mm and continuing until 3 days after the end of an interovulatory interval. Blood samples were drawn by jugular venipuncture into heparinized tubes and immediately stored on ice for transport to the laboratory. After centrifugation, plasma was collected and aliquots were made and stored frozen (-20°C). A heterologous radioimmunoassay previously validated to measure inhibin in rat plasma1,2° was used to measure irinhibin in mare plasma and follicular fluid. The primary antiserum was raised in rabbits against a synthetic porcine inhibin ot-subunit [(1-26)-Gly-Tyr] fragment. The same synthetic peptide was used for preparation of standards and tracer. Various volumes of pooled plasma from 3 mares each during diestrus and estrus and equine follicular fluid (aspirated from a 38 mm follicle during estrus) were assayed in triplicate to check for parallelism against the porcine inhibin c¢-subunit standard curve and the charcoal-extracted porcine follicular fluid dose-response curve. Circulating concenlrations of irinhibin were expressed as pg/ml relative to the porcine inhibin msubunit standard curve. The sensitivity of the assay was 50 pg/ml of plasma. The intra- and inter-assay coefficients of variation were 3% and 10%,respectively. Circulating concentrations of FSH were determined using a heterologous radioimmunoassay previously validated in this laboratory. 8 The intra- and inter-assay coefficients of variation were 15% and 17%, respectively. For determination of circulating concentrations ir-inhibin and FSH, plasma samples were thawed EQUINE VETERINARY S C I E N C E
once and assayed in duplicate with 200 ~1 of plasma/assay tube. Each of the 12 interovulatory intervals were normalized to the mean length of the interovulatory interval (22.2 days; range, 19 to 26) as described) 6Four and 5 mares had intervals longer and shorter, respectively, than the mean; less than 6% of the observations were subjected to the randomization procedure associated with normal iration. In addition, data for 3 days before the first ovulation and 3 days after the second ovulation were added to the interovulatory intervals. Slopes of the dose-response curves for pooled mare plasma, equine and porcine follicular fluid, and the porcine inhibin ct-subunit standard curve were analyzed for parallelism using a factorial analysis of variance. The factors were dose and group (plasma, follicular fluid, and inhibin standard). The slopes were determined to be parallel if the interaction between the indicated factors was not significant (1>>0.05). The Statistical Analysis System General Linear Models procedure for repeated measures was used to determine the effect of day on circulating concentrations of irinhibin and FSH during the interovulatory interval and the 3 days before and 3 days after the interval. Differences between means for concennations of ir-inhibin or FSH were considered significant if the difference exceeded the magnitude of the Least Significant Difference. Pearson's correlation coefficient was used to examine the relationship between mean concentrations of ir-inhibin and FSH during the interovulatory interval and the 3 days before and 3 days after the interval.
RESULTS
Slopes of the dose-response curves for pooled estrus and diestrus mare plasma and equine follicular fluid were similar to the slopes for the porcine inhibin ct-subunit standard curve and the porcine follicular fluid dose-response curve (Fig. 1). All 12 mares had a single ovulation at the beginning of the interval and 3 of 12 had a double ovulation at the end; all double ovulations were synchronous and unilateral. Mares were not subgrouped according to the number of ovulations. Circulating concentrations ofir-inhibin andFSH are shown in Figure 2. There was an effect of day for concentrations of irinhibin (P<0.001) and FSH (P<0.006); least significantdifference (lsd; 1:'<0.05) bars are indicated. Mean circulating concentrations of ir-inhibin and FSH during the interovulatory interval including 3 days before and 3 days after the interval were inversely related (r=--0.548; P<0.002).
DISCUSSION
Concennations ofir-inhibin in equine follicular fluid and mare plasma have not been reported previously. However, in immunoblot analysis the presence of the inhibin a-subunit in equine granulosa-theca cell tumor-conditioned media~7 has
Volume 11, Number 6, 1991
been determined using the same primary antibody reported herein. Furthermore, ir-inhibin has been determined in equine testicular homogenate by displacement of radioiodinated bovine 32 kDa inhibin in a dose-dependent manner) 1 In the present study, the percentage of radioiodinated porcine inhibin a-subunit peptide bound to the primary antibody decreased in a dose-dependent manner as the volume of mare plasma and equine follicular fluid increased. The dose-dependent decrease was parallel to the porcine inhibin a-subunit standard curve and the porcine follicular fluid dose-response curve. The results established that a heterologous radioimmunoassay, previously developed to measure inhibin in rat plasma1.20 can be used to measure changes of peripheral concennations or ir-inhibin in the mare. Identification of "free" monomeric ct inhibin has been reportedla in bovine follicular fluid and serum. The "free" monomeric form of ct inhibin lacked inhibin-like bioactivity but was highly reactive with antisera raised against either 32 kDa inhibin or a synthetic ct-subunit fragment. The immunoassay used in the present study utilized an antiserum raised against a synthetic porcine inhibin a-subunit [(1-26)-GlyTyr] fragment. Potentially, therefore, this assay could be detecting "free" monomeric (ct inhibin) as well as "intact" dimeric forms of inhibin. Additional study is needed to identify and differentiate between "free" monomeric versus "intact" dimeric inhibin in equine follicular fluid and plasma. The mean profiles for both ir-inhibin and FS H were poor representations of the fluctuations in individual mares as previously reported for FSH.° Blood sampling more frequently than once daily will be required to further clarify the association between these 2 hormones. Nevertheless, the profile of mean circulating concennations of FSH in the present study was similar to that reported previously during the estrous cycle in horse mares) 5 Mean daily concennations increased during late estms-early diestms, reached high levels during mid-diestrus, and decreased during late diestrus. Significantchanges in mean concentrations ofir-inhibin andFSH were as follows: 1) ir-inhibin decreased between Days 0 and 1, whereas FSH increased between Days 0 and 5; and 2) irinhibin increased between Days 7 and 12, whereas FSH decreased between Days 11 and 14. Mean circulating concennations of ir-inhibin and FSH were inversely related (significant negative correlation). Circulating concentrations of inhibin during the estrous cycle of other domestic species have been reported to be inversely related during the follicular phase in 2 sows 12 and 5 cows: 8 before and after the FSH/LH surge in 1 cow 12and in 8 cows,6 and during both the follicular and luteal phases in 4 ewes: Circulating concentrations of inhibin in cows6:2.1a and ewes z appeared high around the day of ovulation, whereas in sows 12 concentrations appeared low. In the present study, highest mean concentrations ofplasmair-inhibin occurred on the day of ovulation at both the first and second ovulation of the interovulatory interval. Concentration of ir-inhibin in equine follicular fluid was approximately 1,000-fold higher
321
than in peripheral plasma during estms. Perhaps the high plasma concentrations of ir-inhibin on the day of ovulation were associated, in part, with an increased systemic uptake as the vasculature of the dominant follicle increased; in addition, some of the follicular fluid evacuated at the time of ovulation may have entered the peritoneal cavity19 followed by entry of inhibin into the systemic circulation. Mean circulating concentrations of ir-inhibin significantly decreased 1 day before the second ovulation, but a similar decrease did not occur 1 day before the first ovulation. The cause of the decrease in irinhibin prior to the second ovulation is unknown. Three of the 12 mares had a double ovulation at the end of the interovulatory interval. Because of the small number of observations, a statistical comparison of circulating concentrations of ir-inhibin between mares that had a double and single ovulation was not done. Presumably, mares with more than 1 dominant, viable follicle prior to ovulation would produce more ir-inhibin and hence have higher peripheral concentrations. However, inspection of ir-inhibin plasma concentrations 3 days before, on the day of, and 3 days after the second ovulation did not appear to indicate that concentrations were higher in mares that had a double ovulation versus those that had a single ovulation. It has been reported ~ that viable equine follicles have more bioactive inhibin-like activity than alIetic follicles. In addition, bioactive inhibin-like activity was greater in equine follicular fluid from preovulatory follicles than in follicular fluid from follicles during diestrus and estms.14 It is not known if the bioactive inhibin-like component of follicular fluid from the previous studies is similar to the ir-inhibin assayed in follicular fluid and plasma in the present study.
9. Ginther OJ: Reproductive biology of the mare: Basic and applied aspects. Equiservices, Cross Plains, Wisconsin, 1979. 10. Ginther OJ: Ultrasonic imaging and reproductive events in the mare. Equiservices, Cross Plains, Wisconsin, 1986. 11, Hamada T, Watanabe G, Kokuho T, Taya K, Sasamoto S, Hasegawa Y, Miyamoto K, Igarashi M: Radioimmunoassay of inhibin in various mammals. J Endocrin 122:697-704,1989. 12. Hasegawa Y, Miyamoto K, Igarashi M, Yanaka T, Sasaki K, Iwamura S: Changes in serum concentrations of inhibin during the estrous cycle of the rat, pig, and cow. In Inhibin non-steroidal regulation of follicle stimulating hormone secretion. Burger HG, de Kretser DM, Findlay JK, Igarashi M (eds). Serono Symposia, Academic Press:New York, 42:119-133,1987. 13. Knight PG: Identification and purification of inhibin and inhibin-related proteins. J Reprod Fert, Suppl 43:111-123,1991. 14. Miller KF, Wesson JA, Ginther OJ: Changes in concentrations of circulating gonadotropins following administration of equine follicular fluid to ovariectomized mares. Biol Reprod 21:867872,1979. 15. Miller KF, Berg SL, Sharp DC, Ginther OJ: Concentrations of circulating gonadotropins during various reproductive states in mares. Biol Reprod 22:744-750,1980. 16. Pierson RA, Ginther OJ: Follicular population dynamics during the estrous cycle of the mare. Anita Reprod Sci 14:219231,1987. 17. Piquette GN, Kenney RM, Sertich PL, Yamoto M, Hsueh AJW: Equine granulosa-theca ceUtumors express inhibin a- and 8Asubunit messenger ribonucleic acids and proteins. Biol Reprod 43:1050-1057,1990. 18. Taya K, Kaneko H, Watanabe G, Sasamoto S: In hibin and secretion of FSH in oestrous cycles of cows and pigs. J Reprod Fert, Supp143:151-162,1991. 19. Townson DH, Gin ther OJ: Duration and pattern of follicular evacuation during ovulation in the mare. Anita Reprod Sci 15:131-138,1987. 20. Vaughan JM, Rivier J, Corrigan AZ, McClintock R, Campen CA, Jolley D, Voglmayr JK, Bardin CW, Rivier C, Vale W: Detection and purification of inhibin using antisera generated against synthetic peptide fragments. In Methods in Enzymology, Hormone Action, Part K, Neuroendocrine Peptides. Conn PM (ed). Academic Press: Orlando, FL 168:588-617,1989.
REFERENCES 1. Ackland JF, D'Agostino J, Ringstrom SJ, Hostetler JP, Mann BG, Schwartz NB: Circulating radioimmunoassayable inhibin during periods of transient follicle-stimulating hormone rise: secondary surge and unilateral ovariectomy. Biol Reprod 43:347352,1990. 2. Burger HG: Letter to the editor. Inhibin: Definition and nomenclature, including related substances. Endocrin 122:17011702,1988. 3. Channing CP, BattaSK, Conden W, Ganjam VK, Kenney RM: Levels of inhibin activity and an atretogenic factor(s) in follicular fluid harvested from viable and atretic mare follicles. In Dynamics of ovarian function. Schwartz NB, Hunzicher-Dunn M (eds). Raven Press:New York, pp.73-78,1981. 4. de Jong FH: Inhibin. PhysiolRev 68:555-607,1988. 5. de Kretser DM, Robertson DM: The isolation and physiology of inhibin and related proteins. Biol Reprod 40:33-47,1989. 6. Echternkamp SE, Schanbacher BD: Relationships among circulating concentrations of inhibin, FSH, and LH in cattle at estrus. (Abstract) Therio 33:222,1990. 7. Findlay JK, Clarke IJ, Robertson DM: Inhibin concentrations in ovarian and jugular venous plasma and the relationship of inhibin with follicle-stimulating hormone and luteinizing hormone during the ovine estrus cycle. Endocrin 126:528-535,1990. 8. Freedman LJ, Garcia MC, Ginther OJ: Influence of photoperiod and ovaries on seasonal reproductive activity in mares. Biol Reprod 20:567-574,1979.
322
EQUINE VETERINARY SCIENCE
~!i i. . . .
.~#iii~1%iiiiii, ~!!i!iii~::~,iiii ii:
.....,.41ii!Iii~ '!,J? ....
Refereed
CIRCULATINGCONCENTRATIONSOF IMMUNOREACTIVE INHIBINAND FSH DURINGTHE ESTROUSCYCLEOF MARES D. R. Bergfelt, MS;1 B. G. Mann, BS; 2 N. B. Schwartz, PhD; 2 and O. J. Ginther, VMD, PhD 1
SUMMARY
A heterologous radioimmunoassay developed to measure inhibin in rat plasma was validated and used to characterize changes in peripheral concentrations of immunoreactive inhibin (ir-inhibin) in relation to follicle stimulating hormone (FSH) concentrations during the estrous cycle of mares. The primary antiserum used in the assay was developed against a synthetic porcine inhibin ct-subunit [(1-26)-Gly-Tyr] fragment. The same synthetic peptide was used for preparation of standards and tracer. Slopes of the dose-response curves for pooled estrus and diestrus mare plasma and equine follicular fluid were similar to the slopes for the porcine inhibin t~subunit standard curve and porcine follicular fluid doseresponse curve. Twelve mares were bled once daily beginning when diameter of the largest follicle reached >_25 mm and continuing until 3 days after the end of an interovulatory interval (ovulation=Day 0). Each of the 12 interovulatory intervals were normalized to the mean length of the interovulatory interval (22.2 days; range, 19 to 26). There was an effect Authors' addresses: 1 Department of Veterinary Science, University of Wisconsin-Madison, Madison, Wisconsin 53706; 2Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208. Send reprint requests to Dr. Ginther. Acknowledgments: Supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and Equiculture, Inc., Cross Plains, Wisconsin. The authors thank L. Kulick for preparation of figures, M. Westphal for manuscript preparation, and Drs, C. Rivier and W. Vale for the antigen and antiserum used in the inhibin assay.
Volume 11, Number 6, 1991
of day for concentrations of ir-inhibin (P<0.001) and FSH (P<0.006). Significant mean changes were as follows: 1) irinhibin decreased between Days 0 and 1, whereas FSH increased between Days 0 and 5; and 2) ir-inhibin increased between Days 7 and 12, whereas FSH decreased between Days 11 and 14. Mean concentrations of ir-inhibin and FSH were negatively correlated (r=--0.548; P<0.002). In conclusion, mean peripheral concentrations of ir-inhibin and FSH were inversely related during the estrous cycle of mares.
INTRODUCTION
Follicular fluid of domestic species (porcine, bovine, ovine) contains a component termed inhibin that has the capacity to preferentially suppress follicle stimulating hormone (FSH).4,5 Inhibin has been defined as a glycoprotein hormone consisting of an t~-subunit and 1 of 2 distinct lsubunits, 8k or I~. 2The complete amino acid sequence of the precursor of porcine, bovine, rat, and human inhibin has been predicted from complementary DNA sequences, resulting in the development of sensitive radioimmunoassays.4'5 An antiserum directed against a syntheticporcine inhibin t~-subunit fragment has been used to measure levels of inhibin in rat plasma.l.20 Circulating concentrations of inhibin have been characterized during the estrous cycle in a limited number of sows,12 cows,6.12.18and ewes.7 Circulating concentrations of inhibin in the mare have not been reported. However, immu-
319
.0125 I 100"
Log scale follicular fluid vol ~1) ,125 .5 2 I I I
8 I 8'
o.
'=" B:
~k~
E
Porcinefollicularfluid
T
.
[
llsd
~'~
j
6"
80-
c
FS. 1
1
f 180
~
160
=~-4 "r"
60 Equine foll~lar luid
"~,~.
- 140
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2'
+ o~
ov
2o Porcine inhibin
O'
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~5 =_ Oo
Log scale plasma vol (p.l) lOO 2oo
10o-
~
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,o-
=E
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o.-subunit
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1~0
Figure 1. Radioimmunoassay dose-response curves for pooled estrus and diestrus mare plasma and equine and porcine follicular fluid in relation to the porcine inhibin c¢subunit [(1-26)-Gly-Tyr] standard curve. The slopes of the equine plasma and follicular fluid, between 20 and 90% bound, were not different (P>0.05) from the slopes of the porcine inhibin c~-subunit standard curve and the porcine follicular fluid dose-response curve. noblot results from tumor-conditioned media indicated that inhibin c¢-and I]A-subunitsare produced by equine granulosatheca cell tumors.1z Aproteinaceous FSH-inhibiting component in equine follicularfluid has been reported,3,14but it is not known if it is similar to the inhibin component of porcine, bovine, and ovine follicular fluid. The objectives of this study were to validate an inhibin assay for mares using a heterologous radioimmunoassay developed for rat plasma1,2° and to characterize changes in peripheral concentrations of immunoreactive inhibin (ir-inhibin) in relation to FSH concentrations during the eslxous cycle of mares.
MATERIALS
AND METHODS
Twelve riding-type horse mares 4 to 16 years of age and weighing 400 to 550 kg were used during May to August. Breeds were not known, but appeared to be primarily Quarter Horse and Appaloosa based on size, conformation, and color pattems. Transrectal ultrasonic examination of the ovaries was done as described10 to determine the day of ovulation at 320
,
.
,
,
~
.4 -2 0
.
,
.
,
.
,
,
,
.
,
.
,
.
,
,
,
.
,
.
,
.
~
2 4 s 8 10 12 14 18 18 20 0 N u m b e r of d a y s from ovulation
.
o
2
100
.
4
Figure 2. Mean (_+sem;n=l 2) circulating concentrations of irinhibin and FSH during the interovulatory interval in mares. Ovulation (OV) is Day O. There was an effect of day for both ir-inhibin (P
each end of an interovulatory interval (interval between the ovulations of successive ovulatory periods). If double asynchronous ovulations occurred during a given ovulatory period, the first ovulation was used to assign a beginning and end to the interovulatory interval. Mares were examined and bled once daily beginning when diameter of the largest follicle reached >_25mm and continuing until 3 days after the end of an interovulatory interval. Blood samples were drawn by jugular venipuncture into heparinized tubes and immediately stored on ice for transport to the laboratory. After centrifugation, plasma was collected and aliquots were made and stored frozen (-20°C). A heterologous radioimmunoassay previously validated to measure inhibin in rat plasma1,2° was used to measure irinhibin in mare plasma and follicular fluid. The primary antiserum was raised in rabbits against a synthetic porcine inhibin ot-subunit [(1-26)-Gly-Tyr] fragment. The same synthetic peptide was used for preparation of standards and tracer. Various volumes of pooled plasma from 3 mares each during diestrus and estrus and equine follicular fluid (aspirated from a 38 mm follicle during estrus) were assayed in triplicate to check for parallelism against the porcine inhibin c¢-subunit standard curve and the charcoal-extracted porcine follicular fluid dose-response curve. Circulating concenlrations of irinhibin were expressed as pg/ml relative to the porcine inhibin msubunit standard curve. The sensitivity of the assay was 50 pg/ml of plasma. The intra- and inter-assay coefficients of variation were 3% and 10%,respectively. Circulating concentrations of FSH were determined using a heterologous radioimmunoassay previously validated in this laboratory. 8 The intra- and inter-assay coefficients of variation were 15% and 17%, respectively. For determination of circulating concentrations ir-inhibin and FSH, plasma samples were thawed EQUINE VETERINARY S C I E N C E
once and assayed in duplicate with 200 ~1 of plasma/assay tube. Each of the 12 interovulatory intervals were normalized to the mean length of the interovulatory interval (22.2 days; range, 19 to 26) as described) 6Four and 5 mares had intervals longer and shorter, respectively, than the mean; less than 6% of the observations were subjected to the randomization procedure associated with normal iration. In addition, data for 3 days before the first ovulation and 3 days after the second ovulation were added to the interovulatory intervals. Slopes of the dose-response curves for pooled mare plasma, equine and porcine follicular fluid, and the porcine inhibin ct-subunit standard curve were analyzed for parallelism using a factorial analysis of variance. The factors were dose and group (plasma, follicular fluid, and inhibin standard). The slopes were determined to be parallel if the interaction between the indicated factors was not significant (1>>0.05). The Statistical Analysis System General Linear Models procedure for repeated measures was used to determine the effect of day on circulating concentrations of irinhibin and FSH during the interovulatory interval and the 3 days before and 3 days after the interval. Differences between means for concennations of ir-inhibin or FSH were considered significant if the difference exceeded the magnitude of the Least Significant Difference. Pearson's correlation coefficient was used to examine the relationship between mean concentrations of ir-inhibin and FSH during the interovulatory interval and the 3 days before and 3 days after the interval.
RESULTS
Slopes of the dose-response curves for pooled estrus and diestrus mare plasma and equine follicular fluid were similar to the slopes for the porcine inhibin ct-subunit standard curve and the porcine follicular fluid dose-response curve (Fig. 1). All 12 mares had a single ovulation at the beginning of the interval and 3 of 12 had a double ovulation at the end; all double ovulations were synchronous and unilateral. Mares were not subgrouped according to the number of ovulations. Circulating concentrations ofir-inhibin andFSH are shown in Figure 2. There was an effect of day for concentrations of irinhibin (P<0.001) and FSH (P<0.006); least significantdifference (lsd; 1:'<0.05) bars are indicated. Mean circulating concentrations of ir-inhibin and FSH during the interovulatory interval including 3 days before and 3 days after the interval were inversely related (r=--0.548; P<0.002).
DISCUSSION
Concennations ofir-inhibin in equine follicular fluid and mare plasma have not been reported previously. However, in immunoblot analysis the presence of the inhibin a-subunit in equine granulosa-theca cell tumor-conditioned media~7 has
Volume 11, Number 6, 1991
been determined using the same primary antibody reported herein. Furthermore, ir-inhibin has been determined in equine testicular homogenate by displacement of radioiodinated bovine 32 kDa inhibin in a dose-dependent manner) 1 In the present study, the percentage of radioiodinated porcine inhibin a-subunit peptide bound to the primary antibody decreased in a dose-dependent manner as the volume of mare plasma and equine follicular fluid increased. The dose-dependent decrease was parallel to the porcine inhibin a-subunit standard curve and the porcine follicular fluid dose-response curve. The results established that a heterologous radioimmunoassay, previously developed to measure inhibin in rat plasma1.20 can be used to measure changes of peripheral concennations or ir-inhibin in the mare. Identification of "free" monomeric ct inhibin has been reportedla in bovine follicular fluid and serum. The "free" monomeric form of ct inhibin lacked inhibin-like bioactivity but was highly reactive with antisera raised against either 32 kDa inhibin or a synthetic ct-subunit fragment. The immunoassay used in the present study utilized an antiserum raised against a synthetic porcine inhibin a-subunit [(1-26)-GlyTyr] fragment. Potentially, therefore, this assay could be detecting "free" monomeric (ct inhibin) as well as "intact" dimeric forms of inhibin. Additional study is needed to identify and differentiate between "free" monomeric versus "intact" dimeric inhibin in equine follicular fluid and plasma. The mean profiles for both ir-inhibin and FS H were poor representations of the fluctuations in individual mares as previously reported for FSH.° Blood sampling more frequently than once daily will be required to further clarify the association between these 2 hormones. Nevertheless, the profile of mean circulating concennations of FSH in the present study was similar to that reported previously during the estrous cycle in horse mares) 5 Mean daily concennations increased during late estms-early diestms, reached high levels during mid-diestrus, and decreased during late diestrus. Significantchanges in mean concentrations ofir-inhibin andFSH were as follows: 1) ir-inhibin decreased between Days 0 and 1, whereas FSH increased between Days 0 and 5; and 2) irinhibin increased between Days 7 and 12, whereas FSH decreased between Days 11 and 14. Mean circulating concennations of ir-inhibin and FSH were inversely related (significant negative correlation). Circulating concentrations of inhibin during the estrous cycle of other domestic species have been reported to be inversely related during the follicular phase in 2 sows 12 and 5 cows: 8 before and after the FSH/LH surge in 1 cow 12and in 8 cows,6 and during both the follicular and luteal phases in 4 ewes: Circulating concentrations of inhibin in cows6:2.1a and ewes z appeared high around the day of ovulation, whereas in sows 12 concentrations appeared low. In the present study, highest mean concentrations ofplasmair-inhibin occurred on the day of ovulation at both the first and second ovulation of the interovulatory interval. Concentration of ir-inhibin in equine follicular fluid was approximately 1,000-fold higher
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than in peripheral plasma during estms. Perhaps the high plasma concentrations of ir-inhibin on the day of ovulation were associated, in part, with an increased systemic uptake as the vasculature of the dominant follicle increased; in addition, some of the follicular fluid evacuated at the time of ovulation may have entered the peritoneal cavity19 followed by entry of inhibin into the systemic circulation. Mean circulating concentrations of ir-inhibin significantly decreased 1 day before the second ovulation, but a similar decrease did not occur 1 day before the first ovulation. The cause of the decrease in irinhibin prior to the second ovulation is unknown. Three of the 12 mares had a double ovulation at the end of the interovulatory interval. Because of the small number of observations, a statistical comparison of circulating concentrations of ir-inhibin between mares that had a double and single ovulation was not done. Presumably, mares with more than 1 dominant, viable follicle prior to ovulation would produce more ir-inhibin and hence have higher peripheral concentrations. However, inspection of ir-inhibin plasma concentrations 3 days before, on the day of, and 3 days after the second ovulation did not appear to indicate that concentrations were higher in mares that had a double ovulation versus those that had a single ovulation. It has been reported ~ that viable equine follicles have more bioactive inhibin-like activity than alIetic follicles. In addition, bioactive inhibin-like activity was greater in equine follicular fluid from preovulatory follicles than in follicular fluid from follicles during diestrus and estms.14 It is not known if the bioactive inhibin-like component of follicular fluid from the previous studies is similar to the ir-inhibin assayed in follicular fluid and plasma in the present study.
9. Ginther OJ: Reproductive biology of the mare: Basic and applied aspects. Equiservices, Cross Plains, Wisconsin, 1979. 10. Ginther OJ: Ultrasonic imaging and reproductive events in the mare. Equiservices, Cross Plains, Wisconsin, 1986. 11, Hamada T, Watanabe G, Kokuho T, Taya K, Sasamoto S, Hasegawa Y, Miyamoto K, Igarashi M: Radioimmunoassay of inhibin in various mammals. J Endocrin 122:697-704,1989. 12. Hasegawa Y, Miyamoto K, Igarashi M, Yanaka T, Sasaki K, Iwamura S: Changes in serum concentrations of inhibin during the estrous cycle of the rat, pig, and cow. In Inhibin non-steroidal regulation of follicle stimulating hormone secretion. Burger HG, de Kretser DM, Findlay JK, Igarashi M (eds). Serono Symposia, Academic Press:New York, 42:119-133,1987. 13. Knight PG: Identification and purification of inhibin and inhibin-related proteins. J Reprod Fert, Suppl 43:111-123,1991. 14. Miller KF, Wesson JA, Ginther OJ: Changes in concentrations of circulating gonadotropins following administration of equine follicular fluid to ovariectomized mares. Biol Reprod 21:867872,1979. 15. Miller KF, Berg SL, Sharp DC, Ginther OJ: Concentrations of circulating gonadotropins during various reproductive states in mares. Biol Reprod 22:744-750,1980. 16. Pierson RA, Ginther OJ: Follicular population dynamics during the estrous cycle of the mare. Anita Reprod Sci 14:219231,1987. 17. Piquette GN, Kenney RM, Sertich PL, Yamoto M, Hsueh AJW: Equine granulosa-theca ceUtumors express inhibin a- and 8Asubunit messenger ribonucleic acids and proteins. Biol Reprod 43:1050-1057,1990. 18. Taya K, Kaneko H, Watanabe G, Sasamoto S: In hibin and secretion of FSH in oestrous cycles of cows and pigs. J Reprod Fert, Supp143:151-162,1991. 19. Townson DH, Gin ther OJ: Duration and pattern of follicular evacuation during ovulation in the mare. Anita Reprod Sci 15:131-138,1987. 20. Vaughan JM, Rivier J, Corrigan AZ, McClintock R, Campen CA, Jolley D, Voglmayr JK, Bardin CW, Rivier C, Vale W: Detection and purification of inhibin using antisera generated against synthetic peptide fragments. In Methods in Enzymology, Hormone Action, Part K, Neuroendocrine Peptides. Conn PM (ed). Academic Press: Orlando, FL 168:588-617,1989.
REFERENCES 1. Ackland JF, D'Agostino J, Ringstrom SJ, Hostetler JP, Mann BG, Schwartz NB: Circulating radioimmunoassayable inhibin during periods of transient follicle-stimulating hormone rise: secondary surge and unilateral ovariectomy. Biol Reprod 43:347352,1990. 2. Burger HG: Letter to the editor. Inhibin: Definition and nomenclature, including related substances. Endocrin 122:17011702,1988. 3. Channing CP, BattaSK, Conden W, Ganjam VK, Kenney RM: Levels of inhibin activity and an atretogenic factor(s) in follicular fluid harvested from viable and atretic mare follicles. In Dynamics of ovarian function. Schwartz NB, Hunzicher-Dunn M (eds). Raven Press:New York, pp.73-78,1981. 4. de Jong FH: Inhibin. PhysiolRev 68:555-607,1988. 5. de Kretser DM, Robertson DM: The isolation and physiology of inhibin and related proteins. Biol Reprod 40:33-47,1989. 6. Echternkamp SE, Schanbacher BD: Relationships among circulating concentrations of inhibin, FSH, and LH in cattle at estrus. (Abstract) Therio 33:222,1990. 7. Findlay JK, Clarke IJ, Robertson DM: Inhibin concentrations in ovarian and jugular venous plasma and the relationship of inhibin with follicle-stimulating hormone and luteinizing hormone during the ovine estrus cycle. Endocrin 126:528-535,1990. 8. Freedman LJ, Garcia MC, Ginther OJ: Influence of photoperiod and ovaries on seasonal reproductive activity in mares. Biol Reprod 20:567-574,1979.
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