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Inhibin immunization for increasing ovulation rate and superovulation
Theriogenology41:3-17, 1994. INHIBIN IMMUNIZATION FOR INCREASING OVULATION RATE AND SUPEROVULATION T O'Sheal M A Hillard2, S T AndersonL B M Bindon2, J K Findlay3, C G Tsonis4 and J F Wilkins5 1Department of Physiology, University of New England, Armidale, NSW 2351, Australia; 2CSIRO Division of Animal Production, Armidale, NSW 2350, Australia; 3Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia; 4Biotech Australia Pty Ltd, PO Box 20, East Roseville, NSW 2069, Australia; 5NSW Agriculture, Agricultural Research Station, Grafton, NSW 2460, Australia ABSTRACT The paper reviews the role of inhibin in the ruminant estrous cycle and the potential of inhibin vaccines in sheep and cattle to increase ovulation rate, fecundity and reliability of superovulation in embryo transfer programs. Immunization of ewes with various inhibin preparations (native inhibin, synthetic peptides or recombinant inhibin-a fusion proteins) resulted in an increased ovulation rate, that can be attributed to elevated plasma FSH concentrations. Persistent superovulatory responses were obtained with synthetic peptides. The ovulation rate increases obtained with inhibin immunization and exogenous FSH were shown to be additive so that less exogenous gonadotrophin is required to obtain a given ovulation rate and hence embryo recovery rate. In cattle a prototype vaccine has been developed, based on small doses of recombinant ovine inhibin-ctprotein. This results in raised plasma FSH concentrations and increased ovulation rates after booster vaccinations. Unlike in sheep, synthetic peptide immunization has not, so far, given a satisfactory superovulatory response. Preliminary evidence is reviewed showing that inhibin immunization should also result in less exogenous gonadotrophin being required in embryo transfer programs in cattle. Key words: superovulation, inhibin, immunization,sheep, cattle INTRODUCTION Inhibin was purified to homogeneity in 1985 (45). There followed rapid progress in characterization of the inhibin molecule, its subunit structures and the identification of three inhibin genes (see 15 for review). It is now defined as a glycoprotein hormone consisting of two dissimilar, disulphide-linked subunits, termed a and B, which inhibits the synthesis and/or secretion of the pituitary gonadotrophins, preferentially FSH (7). Two molecular weight forms of bovine inhibin were isolated originally from bovine follicular fluid (44, 45) and two forms have also been described in ovine follicular fluid (28). The structure of bovine (18), porcine (32), ovine (8) and murine (12) inhibins have all been elucidated by cDNA techniques. Three inhibin genes have been identified: the tx gene and the B genes, termed BA and 13B,coding for two forms of inhibin (ctBA, c~BB)and three forms of activin (6ABA, BBBB, BABB) (see 53). The ovarian follicle is the major, and perhaps the only, source of inhibin in the ovine and bovine ovary. Granulosa cells of large antral follicles appear to be the principal site of inhibin et-subunit mRNA expression. There is no evidence of expression of ct- or BA-inhibingenes in either corpora lutea or ovarian stromal tissues of sheep and cattle (see 15 for review). Acknowledgements The authors acknowledge the skilled technical assistance of Yvonne Benton, Faye Hughes, Robert Nethery, Syd Pickering, Darryl Russell and Grant Uphill. The research was supported by grants from the Meat Research Corporation, the Wool Research and Development Corporation and the National Health and Medical Research Council of Australia. We thank NIADDK for the supply of RIA reagents and Dr D M Robertson for provision of purified bovine inhibin.
Copyright © 1994 Butterworth-Heinemann
4
TheHogeno~gy
Activin, consisting of dimeric inhibin B-subunits, has been purified to homogeneity from bovine follicular fluid. In contrast with inhibin, activin stimulates FSH secretion by the pituitary, while, in vitro at least, both inhibin and activin have local paracrine and/or autocrine actions in the ovary (for reviews see 13, 15). Thus, there exists the potential for intricate feedback regulation of both systemic FSH levels and intra-ovarian follicular development by these peptide hormones. There has been increased understanding of the physiology of inhibin as a peripheral hormone controlling FSH secretion, especially in the estrous cycle (e.g., sheep: 14, cattle: 49). It has been postulated that FSH stimulates follicle growth following luteolysis; increasing inhibin and estradiol-17B secretion by the dominant follicle then inhibits FSH synthesis and release by the pituitary, leading to reduced peripheral FSH and subsequent suppression of subordinate follicles from the contemporary cohort (wave) of recruited follicles present at the onset of the follicular phase of the estrous cycle. A similar inverse relationship between peripheral concentrations of FSH and inhibin was observed in cows (49) and after treatment with equine chorionic gonadotrophin (eCG or PMSG) to induce superovulation (27). The above studies provide a sound physiological basis for manipulation of gonadal function by inhibin-specific antibodies. In such studies it is postulated that immunoneutralization of inhibin during the follicular phase will lead to increases in peripheral FSH concentrations and an increase in the number of follicles ovulating. Historically though, the original idea to immunize sheep and cattle against inhibin-related peptides came from the observation that the ovary of the prolific Booroola Merino was deficient in bioactive ovarian inhibin (11). It was postulated that the elevated plasma FSH concentrations and exceptional ovulation rates of the Booroola were a consequence of this inhibin deficiency. VACCINATION AGAINST INHIBINS Three classes of immunogen have been studied-native hormones, synthetic peptides derived from the N terminus of inhibin-et and recombinant inhibins. In the present experiments (Table 1) native inhibins (bMPI and oMPI) were prepared from steroid-free follicular fluid by immunoafflnity chromatography using a monoclonal antibody 256H (34) having strict binding specificity for the c~-subunit of bovine 32 k Da inhibin (40). Synthetic peptide fragments based on the first 32 amino acids of the et-subunit of porcine inhibin and peptide fragments of human and bovine cx-subunits were conjugated to human serum albumin (38). Recombinant bovine, ovine and human ~x-subunit fusion proteins expressed in E. coli (17, 19) have been described for use in vaccination studies. The complete recombinant human inhibin-A molecule has also been investigated. Table 1:
Immunogens used for recent inhibin vaccination studies at Armidale
Description
Abbreviation
Human serum albumin Prepared by immunochromatography from bovine follicular fluid Prepared by immunochromatography from ovine follicular fluid Porcine cxinhibin (1-32) Porcine c~inhibin (1-26) Gly27-Tyr28 Porcine c~inhibin Ser6 (7-26) Gly27-Tyr2S Porcine ¢t inhibin (10-26) Gly27-Tyr28 Porcine et inhibin Ser12 (13-26) Gly27-Tyr2S Porcine ~t inhibin Cys6-TyrT-(8-22)
hSA bMPI oMPI Peptide ~1-32 Peptide o~1_26 Peptide ~7-26 Peptide an0-26 Peptide oq3.26 Peptide ~8-22
Theriogenology
5
Investigations with partially purified inhibins and synthetic inhibin immunogens used Freund's Complete Adjuvant (FCA) for primary immunizing injections (see 40). Studies of recombinant inhibins involved use of Montanide 888:Marcol 52 mixed in the ratio of 1:9 as adjuvant (see 17). Vaccines containing partially purified native inhibin antigens were injected at multiple subcutaneous (n = 4-5) and intramuscular (n= 2) sites, while those containing either synthetic peptides or recombinant inhibins were administered at two intramuscular sites. The period between primary and first booster vaccination (one month) has remained constant. The timing of subsequent booster immunization has varied between studies. Except where indicated, ovarian data were obtained by mid-ventral laparoscopic examination of ovarian structures. Inhibin Vaccination Effects on Ovulation Rate in Sheep and Cattle The first report of increased ovulation rate in sheep following immunization with crude native inhibins was that of O'Shea et al. (41). This was later confirmed (9, 22, 36) and it was also shown that increased prolificacy (i.e., litter size) resulted. In these and other early studies the impurity of the immunogen meant that it was not possible to attribute the effects solely to irnmunoneutralization of inhibin. Once inhibin-A was purified to homogeneity (44) it became possible to test specific inhibin-binding antibody. O'Shea et al. (40) showed that immunization of Merino ewes with native bovine inhibin preparations of increasing purity resulted in parallel increases in specific inhibin-bindingand ovulatory responses. Immunization of ewes against the synthetic peptides based on the NH2-terminal portion of the inhibin-~x chain also significantly increased the ovulation rate two- to four-fold (38, 48). It was shown to be necessary to conjugate the peptides to an immunogenic carrier protein in order to obtain an ovulatory response (33). Wrathall et al. (52) demonstrated that immunization against the bovine synthetic peptide resulted in antibodies which bound labelled 31 kD bovine inhibin, that is to say the native molecule, whereas the other reports describe binding to a labelled peptide but not to inhibin p e r se (33, 38). O'Shea et al. (38) compared the ovarian responses of Merino ewes immunized against native bovine inhibin (i.e., bMPI, as in Table 1; 15 I.tg inhibin in 200 gg protein) or synthetic porcine inhibin ct-peptide. In this study the ewes were immunized on day 0 and 31 of the investigation. Ovulation rates were significantly elevated by day 21 and sustained until day 81. Both immunogens produced significant binding of 125Ihuman inhibin-ct peptide label. Conjugates of smaller synthetic peptide fragments from the N-terminal sequence of inhibin-~x are also effective antigens causing increased ovulation rates. Table 2 shows that amino acids 7-26 are as effective as 1-32 for producing large increases in ovulation rate. Where superovulatory responses were obtained after one or two booster immunizations the increased ovulation rate persisted for at least 13 months (Table 2). This provides a basis for the utilization of immunization to provide for repeated collections of embryos for embryo transfer. In contrast, where the response was less it was more ephemeral and did not persist (e.g., oMPI in Table 2). Immunization of ewes against the recombinant bovine and human m h i b i n - a subunit fusion proteins produced a three- to five-fold increase in ovulation rate. There was serum antibody binding to labelled 31 kD bovine inhibin (17, 50) which correlated with the ovulation rate in individual ewes (50). In contrast to the data with synthetic peptides, conjugation of the bovine fusion protein to a carrier protein was shown to be unnecessary for the response in ovulation rate (17). Immunization against native ovine inhibin prepared by Matrex gel Red A affinity chromatography (10) was shown to increase ovulation rate in mature cows. In this early experiment, 6/12 animals had multiple (two to six) ovulations. Price et al. (43) subsequently described an increase of one to three ovulations in yearling cross-bred heifers immunized with
6
Theriogenology
Table 2:Ovulation rates of Merino ewes immunized against synthetic N-terminal porcine inhibin-ct immunogens (coupled to hSA) or native ovine inhibin (oMPI) (S. Anderson, T. OShea, M. Hillard and B. Bindon, unpublished data) Ovulation rate Antigen hSA al-32 al-26 a7-26 alo-26 ~13-26 as-22 oMPI
OR1
OR2
OR3
OR4
1.8+0.14 1.8+0.11 1.7+0.14 1.7+0.14 1.8+0.11 1.8+0.11 1.6+0.12 1.8+0.11
1.6+0.12 6.3+1.28"* 8.1+1.09"* 7.3+1.27"* 2.8+0.39* 2.8+0.55** 2.3+0.27 3.7+0.8.8**
1.4+0.12 5.2+1.10"* 5.0+0.88** 4.0+0.68** 2.1+0.21 2.2+0.35 1.6+0.17 1.7+0.32
1.9+0.14 6.8+1.09"* 6.0+0.95** 5.3+1.09"* 3.0+0.30* 2.3+0.19 1.7+0.19 2.4+0.54
n = 13 to 17 per group OR1 is pre-immunization ovulation rate OR2 is 18 days after second booster immunization OR3 is 49 days after second booster immunization OR4 is 10 months after second booster immunization * = significantly higher than for control ewes (P < 0.05) from analysis of variance ** = significantly higher than for control ewes (P < 0.01) from analysis of variance a similar extract of ovine follicular fluid. A much more consistent and significant response was obtained (6) when pubertal heifers were immunized with an immunoaffinity-purified preparation of ovine follicular fluid (Table 3). Ovulation response was spectacular but highly variable. Although all immunized heifers show an increased ovulation rate on at least one occasion, the overall repeatability of the response was non-significant. Immunization of mature cattle with synthetic porcine-or peptide (in FCA) has produced variable effects on ovulation rate. In our early investigations about 20% of immunized cows showed two or three ovulations after the first booster immunization (B.M. Bindon, M.A. Hillard and T. O'Shea, unpublished). Similar increases in ovulation rate have been observed in dairy and beef heifers (20, 35, 46, 47). We have now completed more immuno-neutralization studies of heifers using synthetic inhibin-c~ fragments and recombinant inhibin fusion proteins with Montanide:Marcol adjuvant. The results shown in Table 4 can be interpreted as follows: • Synthetic inhibin-c~ fragments were not successful as immunogens to increase ovulation rate, despite evidence of antibody binding (e.g., human Ct|.27 ). • Native ovine inhibin (oMPI) resulted in low inhibin binding but confirmed earlier results by demonstrating large increases in mean ovulation rate. • Recombinant inhibin immunogens led to variable effects on ovulation rate and inhibin binding. Recombinant bovine-or was ineffective despite producing high levels of inhibin binding. Both the complete molecule of human recombinant inhibin and recombinant ovine-~t.3 preparations were effective in increasing ovulation rate. Recombinant ovine inhibin-cc3 (at 500 p.g of fusion protein per immunizing dose) was chosen for evaluation as a vaccine to induce twinning in beef cows. Preliminary fertility and pregnancy rate data (see Table 11, reference 5) are comparable to any previous hormonal manipulation technique used in the bovine.
Theriogenology Table 3:
7
Mean (+SE) ovulation rate and inhibin binding in pubertal beef heifers immunized with partially purified ovine inhibin (oMPI) or ovine serum albumin (oSA) in Freund's Complete Adjuvant (data from reference 6 and unpublished observations of B. Bindon, M. Hillard and T. O'Shea) Days after primary immunization#
Immunogen
n
Observation
57
87
oSA
8
Ovulation rate* 1.0-&0 (3) Animals with OR>I 0 Inhibin binding, <0.1
oMPI
8
Ovulation rate 5.5+3.5 (4) 11.6+3.9 (7) 3.7+1.6 (7) 12.9+4.8 (7) Animals with OR>I 3 7 4 6 Inhibin binding 8.7+1.4 25..0+2.3 29.5+1.7 --
1.0-L-_0(4) 0 <0.1
155
246
1.0!-0 (6) 0 <0.1
1.0+-0 (8) 0 --
# Booster injections on days 25, 65, 129 and 228 after primary * Number of heifers ovulating in parentheses a Percentage L25I-labelled purified oMPI bound by 100 I.tl 1:500 dilution plasma Table 4:
Ovulation rates (OR), number of animals with two or more ovulation rate records of greater than 1 (OR>l) and inhibin binding in heifers after two booster vaccinations with native or synthetic peptides, or recombinant (Rec.) inhibin formulations with Montanide:Marcol (M. Hillard, B. Bindon, J. Wilkins, C. Tsonis and J. Findlay, unpublished data)
OR (mear~+SE)
Heifers with two OR>I
54 16 16 16
1 1 1.1+0.10 1
0 0 0 0
0 0 16+_2 0
8 8 8 8 8 7 8 8
16 24 15 15 15 19 15 15
1.1+0.1 1.7+0.3 1.7+_0.3 1 1.2+0.4 3.4+0.8 2.6+0.1 1.6+_0.3
0 1 2 0 0 5 2 1
33+_3 13+_2 22+3 20+-2 25+3 21+5 25+3 26+3
8 8 8 8
15 21 16 24
1 2.4+0.5 2.4+0.6 7.0+-2.7
0 2 3 3
0 15_+4 16+_5 5+-3
Treatment immunogen
Dose (l.tg)
n
Adjuvant Bovine ~1-27 Human ot1_27 Porcine (7.1.27
-200 200 200
23 8 8 8
500 500 500 1000 2500 500 1000 2500 33.3 100 300 400
Rec. bovine et. 1 Rec. human et.2 Rec. ovine ct.2 Rec. ovine cx.2 Rec. ovine et.2 Rec. ovine et.3 Rec. ovine ~x.3 Rec. ovine (x.3 Rec. human inhibin Rec. human inhibin Rec. human inhibin oMPI
Number of ovulatory cycles
Inhibin* binding (%)
* Inhibin binding refers to percentage 125I-labelled 31 kDa bovine inhibin-A bound by 0.6 ml plasma diluted 1:3.
8
Theriogenology
It is clear from these studies that binding of 31 kDa bovine inhibin to plasma in immunized cattle is not necessarily related to an effect on ovarian function. Conversely, with oMPI quite low levels of specific-inhibin binding were seen, but there were large effects on ovulation rate. It is now known that our cattle inhibin vaccination studies to this point used excessively high doses of inhibin immunogen. A recent, more comprehensive dose response investigation (Table 5) reveals that Rec. ovine inhibin-tx.3 is much more potent than formerly realised. It is clear that doses of immunogen as low as 7.8 I.tg are able to induce multiple ovulation in mature beef females. Further studies are now required to re-assess the inhibin vaccine at low dose levels. Table 5:
Effect of dose of immunogen on ovarian response of multiparous beef cows i m m u n i z e d against recombinant ovine inhibin-ct.3 fusion protein (in Montanide:Marcol) (J. Wilkins, M. Hillard, B. Bindon, C. Tsonis and J. Findlay, unpublished data) Ovulation rate#
Immunogen dose (I.tg)
n
Mean
0 3.9 7.8 15.6 31.2 62.5 500.0
31 18 40 17 40 40 18
1.00 1.00 1.20 1.88 3.08 3.13 3.17
SE
Range
0 0 0.16 0.34 0.45 0.48 0.58
1 1 0-6 1-6 0-15 1-15 1-11
# Ovulation rate measured by real-time ultrasound 30 days after first booster injection given on day 2 of CIRD-PG synchronized cycle Mode of Action of Inhibin Immunization According to our hypothesis, immuno-neutralization of inhibin should lead to a preferential increase in circulating concentration of FSH. In most situations involving native bovine immunogens prepared from follicular fluid and in two independent experiments in which ewes were immunized with recombinant bovine inhibin-tx such an increase in FSH has been confirmed (1, 16, also see 19). In the studies of O'Shea et al. (40) both the degree of inhibin binding and the extent of FSH increase were significantly correlated with the magnitude of ovulation rate increase (r = +0.52, P < 0.001, and r -- +0.39, P < 0.001). There was also a significant correlation between plasma FSH concentration and the coincident specific inhibin binding (r = +0.66, P < 0.001). In the case of sheep immunized against synthetic inhibin-tx peptides variable effects on plasma FSH have been observed (19), despite consistent increases in ovulation rate. This led to suggestions that there may be intra-gonadal effects of inhibin immunization. Recent more intensive blood sampling schedules (3) have shown a transient increase in plasma FSH concentration following primary immunization with peptide 1-32 without any effect on ovulation rate. After a booster immunization an increased ovulation rate was preceded by increased plasma FSH concentration over several days. From this it is inferred that an effective increase in FSH, while not required to be of a large magnitude (24), must be present for some
Theriogenology
9
time to achieve an increase in ovulation rate. Passive immunization of ewes with inhibinbinding antibodies raised against synthetic c~-chainpeptide leads to abrupt increases in plasma FSH concentrations and a three-fold increase in ovulation rate (51). Follicle growth studies using passive immunization (31) support the association between inhibin immunization, plasma FSH concentrations and follicle growth and development. In ewes immunized with peptides mimicking porcine c~-inhibinpeptides (S.T. Anderson, B.M. Bindon, M.A. Hillard and T. O'Shea, unpublished data), antibody binding to l~Ilabelled native 32 kDa inhibin was significantly correlated (n = 76, r = +0.33) with ovulation rate, whereas there was no correlation between ovulation rate and binding to peptide cq-26tracer (n = 76, r = - 0.10). Thus, titres may only be meaningful if measured against the native protein. Heifers immunized with doses of Rec. ovine inhibin-o~.3, varying from 125 I.tg to 1,000 I.tg, show a consistent pattern of inhibin binding (Figure 1), with peak levels evident within seven days of a booster injection. Specific inhibin binding remains elevated for about 14 days, then declines significantly over the next 28 days. Re.boosting promptly induces increased inhibin binding activity. Repeatability of multiple ovulation rate in these inhibin-vaccinated heifers (Table 6) mimics the antibody binding pattern, such that the proportion of cows showing increased ovulation rate declines in the second cycle after booster injection. As in the sheep, plasma FSH concentrations were higher (P<0.05) during the follicular and early luteal phases of the estrous cycle in heifers immunized against Rec. ovine inhibin-c~.3 (Figure 2). In the same study there was no evidence of changes in LH secretion in the immunized females.
Booster 1
Booster 2
40 Level of antigen (Rec. ovine inhibina . 3 /2 ml in vaccine)
30
1000 I,tg 500 I.tg 20 --'-'&
125 I.tg 0 I.tg
10"I
i
250/.tg
i 4
i 8
i 12
i 16
20
W e e k s post-primary immunisation
Figure 1: Inhibin binding in plasma diluted 1:3 in heifers immunized with varying doses of Rec. ovine inhibin-ct.3.
Theriogenology
10
Table 6:
Ovulation rates (OR) in heifers two weeks (synchronised) and five weeks (natural cycle) after boosters 1 and 2 immunization with Rec. ovine inhibin-(z.3 (M. Hillard, B. Bindon, J. Wilkinson, C. Tsonis and J. Findlay, unpublished data) Number with OR >1 Two weeks Five weeks post-booster 1
Treatment group (mg/dose)
Number with OR >1 Two weeks Five weeks post-booster 2
Control
(0)
10
0
0
0
0
I (0.125)
10
5 (2,2,2,3,11)*
1 (2)
4 (2,5,8,1 I)*
1 (3)
2 (0.25
9
5 (2,2,4,8,9)
2 (3,2)
5 (2,3,4,16,37)
2 (2,15)
3 (0.5)
10
6 (2,2,2,3,6,10)
2 (2,2)
5 6 (2,2,2,5,11) (2,2,2,2,8,10)
4 (1.0)
9
4 (2,2,3,3)
2 (2,4)
4 (3,4,5,11)
2 (2,4)
*Individual ORs in brackets.
2.s
.
1"381 i
3
i
|
,
i
21ar0e
i
le y
iati
2G3
•
•
4
to
Figure 2: Plasma FSH before and after prostaglandin (PG) synchronization (10 days postbooster 2) of heifers vaccinated with varying doses (125-1000 gg) of Rec. ovine inhibin-a.3 (see legend Figure 1).
Theriogenology
11
Effects on Puberty Immunization of ewe lambs early in life with inhibins prepared from follicular fluid (2), synthetic inhibin-ct peptides (3, 37) or recombinant bovine-a fusion protein (39) led to some advancement of puberty and increases in ovulation rate (Table 7). It was shown (2, 36) that the effects were more pronounced if the primary immunization was given at age three weeks rather than age nine weeks. In these ewes there were no significant effects of the immunization on plasma FSH or LH concentrations. Table 7:
Effects of immunization of Merino lambs with partially purified bovine inhibin (PPI), recombinant (Rec.ba), synthetic (porcine cq.32) or native (bMPI) inhibin at 3, 7 and 15 weeks of age on inhibin binding (17 weeks) and ovarian activity at 3042 and 72-80 weeks of age (data from 2, 3, 37, 39)
30-42 weeks Immunogen
n
Control PPI
9 9
Control Rec.bcx bMPI
22 25 21
Control Porcine oq.32
12 12
Inhibin binding (%)
Number ovulating
--0.5+0.1 27.2+1.7 12.6+2.3 ---
Ovarian activity 72-80 weeks
OR
Number ovulating
1 6
1.00+0.0 3.33+0.99
9 8
1.00+0.0 2.00+0.57
0 10 3
0 2.95+0.75 2.33+0.88
15 22 15
1.27+0.15 4.41+0.67 1.40+0.16
2 8
1.00+0.0 2.50+0.4
11 11
1.00+0.0 3.10+0.6
OR
In a preliminary study (M.A. Hillard, B.M. Bindon and C.G. Tsonis, unpublished), crossbred beef heifers were immunized with Rec. ovine inhibin-m3 in Montanide:Marcol adjuvant at an average age of 110 days, then boosted 30, 60, 90 and 210 days later. Ovaries , were examined 90 days after the final booster, when the heifers were 14 months old, on average. The data (Table 8) confirm a similar phenomenon to that described in sheep, that is, inhibin vaccination resulted in multiple ovulation with some evidence of advancement of puberty. Table 8:
Treatment
Ovarian activity of beef heifers treated early in life with Rec. ovine inhibin-a.3 and examined at 14 months of age
n
Number with CL or CA
Number of ovulation cycles with OR 1 >1
Control
10
3
4
0
Rec. ovine inhibin-ot.3
11
7
1
10
Ovulation rate (mear~+SE) 1.0+0 3.8+0.5
12
Theriogenology
Inhibin Vaccination and Superovulation for Embryo Transfer Despite the plethora of scientific investigations over the past 15-20 years to refine treatment protocols, variability of superovulation rates and/or numbers of transferable embryos recovered per donor treatment remain as the major limitation to the widespread use of embryo transfer in animal breeding. A reason for this variability is that injection of exogenous gonadotrophins will trigger high peripheral levels of the natural negative feedback regulators (inhibin and estradiol-1713) derived not only from the dominant follicle, but also from the cohort of recruited subordinate follicles which would usually become atretic in an unstimulated estrous cycle (26, 27). The inhibin vaccine approach to superovulation differs in that it is directed at one of these inhibitors that normally counteracts the secretion of endogenous FSH. As a consequence of this approach it is hypothesized that lower dosages of exogenous hormones would be required for superovulation. A dose-response study comparing the ovulatory effects of graded doses of exogenous oFSH(an experimental FSH preparation with 1.8 times the activity of NIH-FSH-S1 and an FSH:LH ratio of 900:1) in control and inhibin-vaccinatedewes (Figure 3) has shown a similar sensitivity of both groups to oFSH treatment (i.e., response slopes parallel), with inhibin vaccination effects on ovulation rate being additive. Thus to attain a set level of superovulation, less exogenous gonadotrophin was required for inhibin-vaccinatedewes.
3-
+
. ~O
O
5..L O
Porcine oq-26
o
Control
O
I
6
b
I
12
Dose of FSH (mg)
Figure 3:
Ovulation rate in control and immunized ewes treated with FSH.
In a preliminary study, Merino ewes were subjected to either a standard PMSG superovulation regime or immunizationwith native bovine inhibin (bMPI 1) or synthetic porcine c~-peptide conjugated to human serum albumin. Embryos were collected surgically and classified by normal embryo assessment standards. The results in Table 9 show significant increases in the number of transferable embryos per synthetic peptide-immunized donor.
Theriogenology Table 9:
13
Ovulation rates and embryo recovery from Merino ewes immunized against inhibins or treated with PMSG (R. Gilchrist, T. O'Shea, G. Hinch and B Bindon, unpublished data)
Immunogen Nil Native bovine Porcine eq.32
n
PMSG
13 11 8
800 iu Nil Nil
Transferable Ovulation Ewes Embryos Percentage embryos rate with per ewe transferable per ewe (meand:SE) embryos (mear~+SE) embryos (mear~+SE) 3.1+0.4 5.0+0.9 11.1+1.9**
10 11 8
1.8+0.4 3.4+0.6 6.4+I .4*
92 65 67
1.7+0.4 2.5+0.6 4.3+1.6*
Porcine eq.32 group significantly different from non-immunized control group ** P < 0.01; * P < 0.05 Preliminary data (23) show that mature Hereford cows immunized against native ovine inhibin (i.e., oMPI with FCA) have increased ovulation rates that resulted in more transferable embryos following mild treatment with exogenous FSH (equivalent to 36 units of NIH oFSH), when compared with non-immunized controls (OR: 16.5+5.6 vs. 2.8+0.5:P<0.05; transferable grade embryos 7.8+2.8 vs. 2.0+0.4: P<0.05). Repeatability of Ovulation Rate in Inhibin-vaccinated Sheep and Cattle In a number of the studies outlined in this review, repeated measures of ovulation rate have been obtained after booster vaccination of ewes or heifers with inhibin antigens. The data have been analysed using least-squares mixed model A N O V A procedures to establish repeatability of ovulation rate. The data in Table 10 show moderate repeatability of ovulation rate in ewes vaccinated with synthetic peptides (particularly tx1.32and tx7.26)and heifers vaccinaTable 10:
Species
Estimates of repeatability of ovulation rates in inhibin-vaccinated sheep and cattle
Antigen
n
OR records per animal
Sheep
Peptide et 1-32 Peptide et 1-26 Peptide ct 7-26 Total e~peptides ~
16 16 15 47
3 3 3 3
Cattle
Rec. o.inhibin.ct.3b oMPIc
16 8
3 or 4 2
Repeatability
P
0.58+0.15 (-)0.07 0.57+0.15 0.41 +0.10
<0.001 NS <0.002 <0.001
0.39+0.16 0.80-2_0.16
<0.003 <0.02
a Post-booster 2 measured through to 10 months (intraclass correlation) b Treated with 500 I.tg fusion protein/vaccination (intraclass correlation) e Superovulatory cycles after booster vacinations 2 and 4, that is, days 87 and 246: Table 3 (repeatability)
14
Theriogenology
ted with 500 I.tg Rec. ovine inhibin-et.3. These estimates are within the range of values (0.40 and 0.69) obtained for prolific Finn sheep and Booroola Merino respectively (42). When the superovulatory cycles of heifers vaccinated with oMPI were analysed, a very high estimate was obtained. This contrasts with the low estimates of repeatability of numbers of ova/embryos (0.23) and transferable embryos (0.15) recovered after superovulation of cattle (21).We know of no comparable data for superovulation in sheep or cattle, despite the perception of industry practitioners that selection of donors with repeatable superovulatory responses is possible. DISCUSSION Knowledge of inhibin biology in ruminants has progressed rapidly since 1985. The availability of pure bovine inhibin- A, synthetic inhibin-ctpeptides and recombinant inhibins has resulted in refinement of early inhibin immunizationstudies and clearer interpretation of the effects observed. Inhibin imrnunogens readily induce increases in ovulation and lambing rates of sheep. The most remarkable effects are: • the increases in ovulation rates after vaccination with quite small peptides (e.g., porcine c~7-26)which persist for more than a year after two booster injections. • the rapid increase in ovulation rate induced by passive immunizationagainst inhibin about the time of luteolysis (51). There is general agreement that vaccination against native and recombinant inhibin causes elevated plasma FSH concentrations in accord with the postulated role of inhibin as a feed-back regulator of FSH synthesis. We have now shown that synthetic inhibin-tx peptide immunogens also act via this pathway. Passive immunization using sera from animals immunized with synthetic peptides (29, 30, 51) confirm that inhibin immunoneutralization induces increased peripheral FSH. Ovulation rate and prolificacy in the cow are more difficult to manipulate by inhibin vaccination. Both native ovine inhibin and recombinant ovine and human inhibins are effective immunogens for inducing increased ovulation rate in the cow. Synthetic peptides have similar effects after multiple booster vaccinations (35). Relatively low levels of 125I-inhibin binding were seen in the cow (i.e., compared to sheep) and some immunogens which result in significant inhibin binding have no effect on ovulation rate. In this review, we provide the first evidence that inhibin immunizationof heifers results in a preferential increase in plasma FSH levels during the follicular phase of the (synchronized) estrous cycle. However, no correlation could be found between the elevated levels of FSH and coincident inhibin binding or subsequent ovulation rate. This may be due principally to the limited (daily) FSI-I concentrations measured. A prototype recombinant ovine inhibin vaccine is currently being evaluated for twinning in cattle. Early fertility and pregnancy results are comparable to previous hormone-based techniques for twinning (4). Recent studies have revealed that the potency of the irnmunogen was far greater than anticipated. Further evaluation may result in an improved technique suitable for commercial application. Inhibin vaccination in the ewe lamb has had variable effects on age at puberty but consistently increased ovulation rate. Early immunizationof the heifer also produces increased ovulation rates with some suggestion of an effect on age at puberty. This aspect requires further research effort. The use of inhibin vaccination holds promise as a means to overcome the variability in superovulation for embryo transfer in sheep and cattle. Preliminary studies outlined in this review reveal that exogenous gonadotrophin treatments can be reduced or eliminated for superovulation in the sheep at least. The recent demonstration (25) that passive inhibin immunoneutralization of cattle on day 12 of the estrous cycle resulted in significant elevation of peripheral FSI-I with coincident increases in the number of small, medium and large ovarian follicles, without the excessive estradiol production observed with exogenous gonadotrophin
Theriogenology
15
treatment (26, 27) supports our contention that inhibin vaccination will improve superovulation regimes. M o d e r a t e to high estimates o f repeatability o f ovulation rates in inhibin vaccinated sheep and heifers also support our hypothesis that less variable repeated superovulation should be achieved with this approach. Future Directions It is essential that any commercial products arising from this technology be acceptable to the general c o m m u n i t y . Further research should be directed at vaccine formulation. In this regard the use o f non-ulcerative aqueous vaccines with novel adjuvants (e.g., s a p o n i n s - Q u i l A, and the i m m u n e s y s t e m cytokines-interleukins and colony stimulating factors) should be investigated. T h e r e is also scope for further investigations o f such aspects as vaccination protocols and i m m u n o g e n dosage using either recombinant or synthetic inhibins. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
AI-Obaidi SAR, Bindon BM, Findlay JK, Hillard MA and O'Shea T. Plasma follicle stimulating hormone in Merino ewes immunized with an inhibin-enriched fraction from bovine follicular fluid. Anim. Reprod. Sci. 1987; 14:39-51. Al-Obaidi SAR, Bindon BM, Hillard MA and O'Shea T. Reproductive characteristics of lambs actively immunized early in life with inhibin-enriched preparations from follicular fluid of cows. J. Reprod. Fertil. 1987; 81:403-414. Anderson ST. 'Inhibin, Peptides and Reproduction in Sheep.' PhD. Thesis, University of New England, Armidale. 1993 Bindon BM and Hillard MA. In: "Twinning in cattle" Cummins LJ. Proc. Aust. Soc. Anim. Prod. 1992; 19:438-447. Bindon BM, Anderson ST, Cummins LJ, Findlay JK, Hillard MA, O'Shea T, Paull D, Tsonis CG and Wilkins JF. Manipulation of reproduction of sheep and cattle by vaccination against inhibin-related peptides. Aust. J. Agric. Res. 1993; in press. Bindon BM, O'Shea T, Miyamoto K, Hillard MA, Piper LR, Nethery RID and Uphill G. Superovulation in pubertal heifers immunized against ovine inhibin purified by monoclonai antibody affinity chromatography. Proc. Aust. Soc. Reprod. Biol. 1988; 20:28 (abstr.). Burger HG. Inhibin: definition and nomenclature, including related substances. J. Endocrinol. 1988; 117:159-160. Crawford RJ, Hammond VE, Evans BA, Coghlan JP, Haralambidis J, Hudson B, Penschow JD, Richards RI and Tregear GW. ¢L-Inhibin gene expression occurs in the ovine adrenal cortex, and is regulated by adrenocorticotropin. Molec. Endocrinol. 1987; 1: 699-706. Cummins LJ, O'Shea T, AI-Obaidi SAR, Bindon BM and Findlay JK. Increase in ovulation rate after immunization of Merino ewes with a fraction of bovine follicular fluid containing inhibin activity. J. Reprod. Fertil. 77:365-372 (1986). Cummins LJ, O'Shea T and Bindon BM. Increased ovulation rates in cattle vaccinated with a partially purified fraction of ovine follicular fluid. Proc. Aust. Soc. Reprod. Biol. 1986; 18:39 (abstr.). Cummins LJ, O'Shea T, Bindon BM, Lee VWK and Findlay JK. Ovarian inhibin content and sensitivity to inhibin in Booroola and control strain Merino ewes. J. Reprod. Fertil. 1983; 67:1-7. Esch FS, Shimasaki S, Cooksey K, Mercado M, Mason AJ, Ying S, Ueno N and Ling N. Complementary Deoxyribonucleic Acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Molec. Endocdnol. 1987; 1: 388-396. Findlay JK. An update on the roles of inhibin, activin and follistatin as local regulators of folliculogenesis. Biol. Reprod. 1993; 48:15-23. Findlay JK, Clarke IJ and 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 estrous cycle. Endocrinology 1990; 126: 528-535. Findlay JK, Clarke IJ, Luck MR, Rodgers RJ, Shukovski L, Robertson DM, Klein R, Murray JF, Scaramuzzi RJ, Bindon BM, O'Shea T, Tsonis CG and Forage RG. Peripheral and intragonadal actions of inhibin-related peptides. J. Reprod. Fertil 1991; 43:Suppl. 139-150.
16
Theriogenology
16. Findlay JK, Doughton B, Robertson DM and Forage RG. Effects of immunization against recombinant bovine inhibin c~subunit on circulating concentrations of gonadotrophins in ewes. J. Endocrinol. 1989; 120:59-65. 17. Forage RG, Brown RW, Oliver KJ, Attache BT, Devine PL, Hudson GC, Goss WH, Bertram KC, Tolstoshev P, Robertson DM, de Kretser DM, Doughton B, Burger HG and Findlay JK. Immunization against an inhibin subunit produced by recombinant DNA techniques results in increased ovulation rate in sheep. J. Endocrinol. 1987; 114:RI-R4. 18. Forage RG, Ring JM, Brown RW, Mclnerney BV, Corbon GS, Gregson RP, Robertson DM, Morgan FJ, Hearn MTW, Findlay JK, Wettenhall REH, Burger HG and de K.retser DM. Cloning and sequence analysis of eDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc. Nat. Acad. Sci. USA 1986; 83:3091-3095. 19. Forage RG, Tsonis CG, Brown RW, Hungerford J'W, Greenwood PE and Findlay JK. Inhibin: a novel fecundity vaccine. In "Animal Health and Production for the 21st Century'. (Ed. Beh KJ) CSIRO, Melbourne. pp. 122-130. 1993. 20. Glencross RG, Bleach ECL, McLeod BJ, Beard AJ and Knight PG. Effect of active immunization of heifers against inhibin on plasma FSH concentration, ovarian follicular development and ovulation rate. J. Endocrinol. 1992; 134:11-18. 21. Hahn J. Attempts to explain and reduce variability of superovulation. Theriogenology 1992; 38:269275. 22. Henderson KM, Franchimont P, Lecomte-Yema MJ, Hudson N and Ball K. Increase in ovulation rate after active immunization of sheep with inhibin partially purified from bovine follicular fluid. J. Endocrinol. 1984; 102:305-309. 23. Hillard MA, Bindon BM, King B, O'Shea T, Andrews CM and Hinch GN. Superovulation of cows immunized against native ovine inhibin. Prec. Aust. Soc. Reprod. Biol. 1990; 22:134 (abstr.). 24. Ireland JJ. Control of follicular growth and development. J. Reprod. Fertil. Suppl. 1987; 34:39-54. 25. Kaneko H, Nakanishi Y, Taya K, Kishi H, Watanabe G, Sasamoto S and Hasegawa Y. Evidence that inhibin is an important factor in regulation of FSH secretion during the mid-luteal phase in cows. J. Endocrinol. 1993; 136:35-41. 26. Kaneko H, Terada T, Watanabe G, Taya K and Sasamoto S. Changes in peripheral levels of immunoreactive inhibin and gonadotrophins in cattle induced to superovulate by porcine folliclestimulating hormone. Jpn. J. Anim. Reprod. 1990; 36:77-82. 27. Kaneko H, Watanabe G, Taya K and Sasamoto S. Changes in peripheral levels of bioactive and immunoreactive inhibin, estradiol-1713, progesterone, luteinizing hormone and follicle stimulating hormone associated with follicular development in cows induced to ovulate with equine chorionic gonadotrophin. Biol. Reprod. 1992; 47:76-82. 28. Leversha LJ, Robertson DM, de Vos FL, Morgan FJ, Hearn MTW, Wettenhall REH, Findlay JK, Burger HG and de Kretser DM. Isolation of inhibin from ovine follicular fluid. 1987; J. Endocrinol. 113:213-221. 29. Mann GE, Campbell BK, McNeilly AS and Baird DT. Passively immunizing ewes against inhibin during the luteal phase of the oestrous cycle raises the plasma concentration of FSH. J. Endocrinol. 1989; 123:383-391. 30. Mann GE, Campbell BK, McNeilly AS and Baird DT. The effects of passively immunizing ewes against inhibin and oestradiol during the follicular phase of the oestrous cycle. J. Endocrinol. 1990 125:417-424. 31. Mann GE, Campbell BK, McNeilly AS and Baird DT. Follicular development and ovarian hormone secretion following passive immunization of ewes against inhibin or oestradiol. J. Endocrinol. 1993 136:225-233. 32. Mason AJ, Hayflick JJ, Ling N, Esch F, Ueno N, Ying SY, Guilleman R, Niall H and Seeburg PH. Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-g. Nature 1985; 318:659-663. 33. Meyer RL, Carlson KM, Rivier J and Wheaton JE. Antiserum to an inhibin alpha-chain peptide neutralizes inhibin bioactivity and increases ovulation rate in sheep. J. Anim. Sci. 1991; 69:747-754. 34. Miyamoto K, Hasegawa Y, Fukuda M and Igarashi M. Demonstration of high molecular weight forms of inhibin in bovine follicular fluid (bFF) by using monoclonal antibodies to bFF 32k inhibin. Biochem. Biophys. Res. Commun. 1986; 136:1103-1109. 35. Morris DG, McDermott MG, Diskin MG, Morrison CA, Swift PJ and Sreenan JM. Effect of immunization against synthetic peptide sequences of bovine inhibin ct-subunit on ovulation rate and twincalving rate in heifers. J. Reprod. Fertil. 1993; 97:255-261.
Theriogenology
17
36. O'Shea T, AI-Obaidi SAR, Bindon BM, Cummins LJ, Findlay JK and Hillard MA Increased ovulation rate in Merino ewes and advancement of puberty in Merino lambs immunized with a preparation enriched in inhibin. In 'Reproduction of Sheep.' (Eds. Lindsay DR and Pearce DT). Australian Academy of Science, Canberra. 1984. pp. 335-337. 37. O'Shea T, Anderson ST, Bindon BM, Hillard MA and Munro RK. Immunization against a synthetic inhibin fragment advances onset of puberty in female Merino lambs. J. Reprod. Fertil. Abstract Series 1989; 4:8 (abstr.). 38. O'Shea T, Andrews CM, Bindon BM, Hillard MA, Miyamoto K and Sinosich MJ. Immunization of Merino ewes with a synthetic inhibin peptide or with preparations obtained from bovine and porcine follicular fluids by immunoaffinity chromatography result in different effects on ovulation rate and on plasma gonadotrophin concentrations. Reprod. Fertil. Dev. 1991; 3:659-670. 39. O'Shea T, Bindon BM, Forage RG, Findlay JK and Tsonis CG. Active immunization of Merino ewe lambs with bovine recombinant alpha inhibin advances puberty and increases ovulation rate. Reprod. Fertil. Dev. 1993; 5:173-180. 40. O'Shea T, Bindon BM, Hillard MA, Piper LR, Findlay JK and Miyamoto K. Increase in ovulation rate in Merino ewes after active immunization with inhibin preparations obtained by immunoaffinity chromatography. Reprod. Fertil. Dev. 1989; 1:347-355. 41. O'Shea T, Cummins LJ, Bindon BM and Findlay JK. Increased ovulation rate in ewes vaccinated with an inhibin enriched fraction from bovine follicular fluid.. Proc. Aust. Soc. Reprod. Biol. 1982; 14:85 (abstr.). 42. Piper LR and Bindon BM. The genetics and endocrinology of the Booroola sheep F gene. In: Proc. Second Int. Conf. Quantitative Genetics (Eds Weir BS, Eissen EJ, Goodman MM and Namkoong G). Sinauer Associates Inc., Sunderland Massachusetts. 1988. pp. 270-280. 43. Price. CA, Morris BA, O'Shea T and Webb R. Active immunization of cattle against partly purified follicular fluid from sheep. 1987; J. Reprod. Fertil. 81:161-168. 44. Robertson DM, de Vos FL, Foulds LM, Burger HG, Morgan FJ, Hearn MTW and de Kretser DM. Isolation of a 31 kDa form of inhibin from bovine follicular fluid. Molec. Cell. Endocrinol. 1986; 44:271-277. 45. Robertson DM, Foulds LM, Leversha L, Morgan FJ, Hearn MTW, Burger HG, Wettenhall REH and de Kretser DM. Isolation of inhibin from bovine follicular fluid. Biochem. Biophys. Res. Commun. 1985; 126:220-226. 46. Scanlon AR, Sunderland S J, Martin TL, Goulding D, O'Callaghan D, Williams DH, Headon DR, Boland MP, Ireland JJ and Roche JF. Active immunization of heifers against a synthetic fragment of bovine inhibin. J. Reprod. Fertil. 1993; 97:213-222. 47. Schanbacher BD. Ovulation response and pituitary FSH charge microheterogeneity in beef heifers vaccinated against synthetic porcine inhibin alpha. J. Dairy Sci. 72:Suppl. 1989; 1,361. 48. Schanbacher BD, Schemm SR and Rhind SM. Gonadotrophin concentrations and ovulation rates in Suffolk ewes actively or passively immunised against inhibin alpha. J. Reprod. Fertil. 1991; 93:133139. 49. Taya K, Kaneko H, Watanabe G and Sasamoto S. Inhibin and secretion of FSH in oestrous cycles of cows and pigs. J. Reprod. Fertil. Suppl. 1991; 43:151-162. 50. Tsonis CG, Pearson M, Hungerford J, Borchers CE, Greenwood PE, Forage RG, Doughton B and Findlay JK. Immunising sheep with recombinant inhibin ct-subunit increases the number Iambs born. Proc. Aust. Soc. Reprod. Biol. 1989; 21:99 (abstr.). 51. Wheaton .rE, Carlson KM and Kusina NT. Active and passive immunoneutralization of inhibin increases follicle-stimulating hormone levels and ovulation rate in ewes. Biol. Reprod. 1992; 47:361-367. 52. Wrathall JHM, McLeod B J, Glencross RG, Beard AJ and Knight PG. Inhibin immunoneutralization by antibodies raised against synthetic peptide sequences of inhibin a-subunit: effects on gonadotrophin concentrations and ovulation rate in sheep. J. Endocrinol. 1990; 124:167-176. 53. Ying SY. Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of folliclestimulating hormone. Endocrine Reviews 1988; 9:267-293.
Copyright © 1994 Butterworth-Heinemann
4
TheHogeno~gy
Activin, consisting of dimeric inhibin B-subunits, has been purified to homogeneity from bovine follicular fluid. In contrast with inhibin, activin stimulates FSH secretion by the pituitary, while, in vitro at least, both inhibin and activin have local paracrine and/or autocrine actions in the ovary (for reviews see 13, 15). Thus, there exists the potential for intricate feedback regulation of both systemic FSH levels and intra-ovarian follicular development by these peptide hormones. There has been increased understanding of the physiology of inhibin as a peripheral hormone controlling FSH secretion, especially in the estrous cycle (e.g., sheep: 14, cattle: 49). It has been postulated that FSH stimulates follicle growth following luteolysis; increasing inhibin and estradiol-17B secretion by the dominant follicle then inhibits FSH synthesis and release by the pituitary, leading to reduced peripheral FSH and subsequent suppression of subordinate follicles from the contemporary cohort (wave) of recruited follicles present at the onset of the follicular phase of the estrous cycle. A similar inverse relationship between peripheral concentrations of FSH and inhibin was observed in cows (49) and after treatment with equine chorionic gonadotrophin (eCG or PMSG) to induce superovulation (27). The above studies provide a sound physiological basis for manipulation of gonadal function by inhibin-specific antibodies. In such studies it is postulated that immunoneutralization of inhibin during the follicular phase will lead to increases in peripheral FSH concentrations and an increase in the number of follicles ovulating. Historically though, the original idea to immunize sheep and cattle against inhibin-related peptides came from the observation that the ovary of the prolific Booroola Merino was deficient in bioactive ovarian inhibin (11). It was postulated that the elevated plasma FSH concentrations and exceptional ovulation rates of the Booroola were a consequence of this inhibin deficiency. VACCINATION AGAINST INHIBINS Three classes of immunogen have been studied-native hormones, synthetic peptides derived from the N terminus of inhibin-et and recombinant inhibins. In the present experiments (Table 1) native inhibins (bMPI and oMPI) were prepared from steroid-free follicular fluid by immunoafflnity chromatography using a monoclonal antibody 256H (34) having strict binding specificity for the c~-subunit of bovine 32 k Da inhibin (40). Synthetic peptide fragments based on the first 32 amino acids of the et-subunit of porcine inhibin and peptide fragments of human and bovine cx-subunits were conjugated to human serum albumin (38). Recombinant bovine, ovine and human ~x-subunit fusion proteins expressed in E. coli (17, 19) have been described for use in vaccination studies. The complete recombinant human inhibin-A molecule has also been investigated. Table 1:
Immunogens used for recent inhibin vaccination studies at Armidale
Description
Abbreviation
Human serum albumin Prepared by immunochromatography from bovine follicular fluid Prepared by immunochromatography from ovine follicular fluid Porcine cxinhibin (1-32) Porcine c~inhibin (1-26) Gly27-Tyr28 Porcine c~inhibin Ser6 (7-26) Gly27-Tyr2S Porcine ¢t inhibin (10-26) Gly27-Tyr28 Porcine et inhibin Ser12 (13-26) Gly27-Tyr2S Porcine ~t inhibin Cys6-TyrT-(8-22)
hSA bMPI oMPI Peptide ~1-32 Peptide o~1_26 Peptide ~7-26 Peptide an0-26 Peptide oq3.26 Peptide ~8-22
Theriogenology
5
Investigations with partially purified inhibins and synthetic inhibin immunogens used Freund's Complete Adjuvant (FCA) for primary immunizing injections (see 40). Studies of recombinant inhibins involved use of Montanide 888:Marcol 52 mixed in the ratio of 1:9 as adjuvant (see 17). Vaccines containing partially purified native inhibin antigens were injected at multiple subcutaneous (n = 4-5) and intramuscular (n= 2) sites, while those containing either synthetic peptides or recombinant inhibins were administered at two intramuscular sites. The period between primary and first booster vaccination (one month) has remained constant. The timing of subsequent booster immunization has varied between studies. Except where indicated, ovarian data were obtained by mid-ventral laparoscopic examination of ovarian structures. Inhibin Vaccination Effects on Ovulation Rate in Sheep and Cattle The first report of increased ovulation rate in sheep following immunization with crude native inhibins was that of O'Shea et al. (41). This was later confirmed (9, 22, 36) and it was also shown that increased prolificacy (i.e., litter size) resulted. In these and other early studies the impurity of the immunogen meant that it was not possible to attribute the effects solely to irnmunoneutralization of inhibin. Once inhibin-A was purified to homogeneity (44) it became possible to test specific inhibin-binding antibody. O'Shea et al. (40) showed that immunization of Merino ewes with native bovine inhibin preparations of increasing purity resulted in parallel increases in specific inhibin-bindingand ovulatory responses. Immunization of ewes against the synthetic peptides based on the NH2-terminal portion of the inhibin-~x chain also significantly increased the ovulation rate two- to four-fold (38, 48). It was shown to be necessary to conjugate the peptides to an immunogenic carrier protein in order to obtain an ovulatory response (33). Wrathall et al. (52) demonstrated that immunization against the bovine synthetic peptide resulted in antibodies which bound labelled 31 kD bovine inhibin, that is to say the native molecule, whereas the other reports describe binding to a labelled peptide but not to inhibin p e r se (33, 38). O'Shea et al. (38) compared the ovarian responses of Merino ewes immunized against native bovine inhibin (i.e., bMPI, as in Table 1; 15 I.tg inhibin in 200 gg protein) or synthetic porcine inhibin ct-peptide. In this study the ewes were immunized on day 0 and 31 of the investigation. Ovulation rates were significantly elevated by day 21 and sustained until day 81. Both immunogens produced significant binding of 125Ihuman inhibin-ct peptide label. Conjugates of smaller synthetic peptide fragments from the N-terminal sequence of inhibin-~x are also effective antigens causing increased ovulation rates. Table 2 shows that amino acids 7-26 are as effective as 1-32 for producing large increases in ovulation rate. Where superovulatory responses were obtained after one or two booster immunizations the increased ovulation rate persisted for at least 13 months (Table 2). This provides a basis for the utilization of immunization to provide for repeated collections of embryos for embryo transfer. In contrast, where the response was less it was more ephemeral and did not persist (e.g., oMPI in Table 2). Immunization of ewes against the recombinant bovine and human m h i b i n - a subunit fusion proteins produced a three- to five-fold increase in ovulation rate. There was serum antibody binding to labelled 31 kD bovine inhibin (17, 50) which correlated with the ovulation rate in individual ewes (50). In contrast to the data with synthetic peptides, conjugation of the bovine fusion protein to a carrier protein was shown to be unnecessary for the response in ovulation rate (17). Immunization against native ovine inhibin prepared by Matrex gel Red A affinity chromatography (10) was shown to increase ovulation rate in mature cows. In this early experiment, 6/12 animals had multiple (two to six) ovulations. Price et al. (43) subsequently described an increase of one to three ovulations in yearling cross-bred heifers immunized with
6
Theriogenology
Table 2:Ovulation rates of Merino ewes immunized against synthetic N-terminal porcine inhibin-ct immunogens (coupled to hSA) or native ovine inhibin (oMPI) (S. Anderson, T. OShea, M. Hillard and B. Bindon, unpublished data) Ovulation rate Antigen hSA al-32 al-26 a7-26 alo-26 ~13-26 as-22 oMPI
OR1
OR2
OR3
OR4
1.8+0.14 1.8+0.11 1.7+0.14 1.7+0.14 1.8+0.11 1.8+0.11 1.6+0.12 1.8+0.11
1.6+0.12 6.3+1.28"* 8.1+1.09"* 7.3+1.27"* 2.8+0.39* 2.8+0.55** 2.3+0.27 3.7+0.8.8**
1.4+0.12 5.2+1.10"* 5.0+0.88** 4.0+0.68** 2.1+0.21 2.2+0.35 1.6+0.17 1.7+0.32
1.9+0.14 6.8+1.09"* 6.0+0.95** 5.3+1.09"* 3.0+0.30* 2.3+0.19 1.7+0.19 2.4+0.54
n = 13 to 17 per group OR1 is pre-immunization ovulation rate OR2 is 18 days after second booster immunization OR3 is 49 days after second booster immunization OR4 is 10 months after second booster immunization * = significantly higher than for control ewes (P < 0.05) from analysis of variance ** = significantly higher than for control ewes (P < 0.01) from analysis of variance a similar extract of ovine follicular fluid. A much more consistent and significant response was obtained (6) when pubertal heifers were immunized with an immunoaffinity-purified preparation of ovine follicular fluid (Table 3). Ovulation response was spectacular but highly variable. Although all immunized heifers show an increased ovulation rate on at least one occasion, the overall repeatability of the response was non-significant. Immunization of mature cattle with synthetic porcine-or peptide (in FCA) has produced variable effects on ovulation rate. In our early investigations about 20% of immunized cows showed two or three ovulations after the first booster immunization (B.M. Bindon, M.A. Hillard and T. O'Shea, unpublished). Similar increases in ovulation rate have been observed in dairy and beef heifers (20, 35, 46, 47). We have now completed more immuno-neutralization studies of heifers using synthetic inhibin-c~ fragments and recombinant inhibin fusion proteins with Montanide:Marcol adjuvant. The results shown in Table 4 can be interpreted as follows: • Synthetic inhibin-c~ fragments were not successful as immunogens to increase ovulation rate, despite evidence of antibody binding (e.g., human Ct|.27 ). • Native ovine inhibin (oMPI) resulted in low inhibin binding but confirmed earlier results by demonstrating large increases in mean ovulation rate. • Recombinant inhibin immunogens led to variable effects on ovulation rate and inhibin binding. Recombinant bovine-or was ineffective despite producing high levels of inhibin binding. Both the complete molecule of human recombinant inhibin and recombinant ovine-~t.3 preparations were effective in increasing ovulation rate. Recombinant ovine inhibin-cc3 (at 500 p.g of fusion protein per immunizing dose) was chosen for evaluation as a vaccine to induce twinning in beef cows. Preliminary fertility and pregnancy rate data (see Table 11, reference 5) are comparable to any previous hormonal manipulation technique used in the bovine.
Theriogenology Table 3:
7
Mean (+SE) ovulation rate and inhibin binding in pubertal beef heifers immunized with partially purified ovine inhibin (oMPI) or ovine serum albumin (oSA) in Freund's Complete Adjuvant (data from reference 6 and unpublished observations of B. Bindon, M. Hillard and T. O'Shea) Days after primary immunization#
Immunogen
n
Observation
57
87
oSA
8
Ovulation rate* 1.0-&0 (3) Animals with OR>I 0 Inhibin binding, <0.1
oMPI
8
Ovulation rate 5.5+3.5 (4) 11.6+3.9 (7) 3.7+1.6 (7) 12.9+4.8 (7) Animals with OR>I 3 7 4 6 Inhibin binding 8.7+1.4 25..0+2.3 29.5+1.7 --
1.0-L-_0(4) 0 <0.1
155
246
1.0!-0 (6) 0 <0.1
1.0+-0 (8) 0 --
# Booster injections on days 25, 65, 129 and 228 after primary * Number of heifers ovulating in parentheses a Percentage L25I-labelled purified oMPI bound by 100 I.tl 1:500 dilution plasma Table 4:
Ovulation rates (OR), number of animals with two or more ovulation rate records of greater than 1 (OR>l) and inhibin binding in heifers after two booster vaccinations with native or synthetic peptides, or recombinant (Rec.) inhibin formulations with Montanide:Marcol (M. Hillard, B. Bindon, J. Wilkins, C. Tsonis and J. Findlay, unpublished data)
OR (mear~+SE)
Heifers with two OR>I
54 16 16 16
1 1 1.1+0.10 1
0 0 0 0
0 0 16+_2 0
8 8 8 8 8 7 8 8
16 24 15 15 15 19 15 15
1.1+0.1 1.7+0.3 1.7+_0.3 1 1.2+0.4 3.4+0.8 2.6+0.1 1.6+_0.3
0 1 2 0 0 5 2 1
33+_3 13+_2 22+3 20+-2 25+3 21+5 25+3 26+3
8 8 8 8
15 21 16 24
1 2.4+0.5 2.4+0.6 7.0+-2.7
0 2 3 3
0 15_+4 16+_5 5+-3
Treatment immunogen
Dose (l.tg)
n
Adjuvant Bovine ~1-27 Human ot1_27 Porcine (7.1.27
-200 200 200
23 8 8 8
500 500 500 1000 2500 500 1000 2500 33.3 100 300 400
Rec. bovine et. 1 Rec. human et.2 Rec. ovine ct.2 Rec. ovine cx.2 Rec. ovine et.2 Rec. ovine et.3 Rec. ovine ~x.3 Rec. ovine (x.3 Rec. human inhibin Rec. human inhibin Rec. human inhibin oMPI
Number of ovulatory cycles
Inhibin* binding (%)
* Inhibin binding refers to percentage 125I-labelled 31 kDa bovine inhibin-A bound by 0.6 ml plasma diluted 1:3.
8
Theriogenology
It is clear from these studies that binding of 31 kDa bovine inhibin to plasma in immunized cattle is not necessarily related to an effect on ovarian function. Conversely, with oMPI quite low levels of specific-inhibin binding were seen, but there were large effects on ovulation rate. It is now known that our cattle inhibin vaccination studies to this point used excessively high doses of inhibin immunogen. A recent, more comprehensive dose response investigation (Table 5) reveals that Rec. ovine inhibin-tx.3 is much more potent than formerly realised. It is clear that doses of immunogen as low as 7.8 I.tg are able to induce multiple ovulation in mature beef females. Further studies are now required to re-assess the inhibin vaccine at low dose levels. Table 5:
Effect of dose of immunogen on ovarian response of multiparous beef cows i m m u n i z e d against recombinant ovine inhibin-ct.3 fusion protein (in Montanide:Marcol) (J. Wilkins, M. Hillard, B. Bindon, C. Tsonis and J. Findlay, unpublished data) Ovulation rate#
Immunogen dose (I.tg)
n
Mean
0 3.9 7.8 15.6 31.2 62.5 500.0
31 18 40 17 40 40 18
1.00 1.00 1.20 1.88 3.08 3.13 3.17
SE
Range
0 0 0.16 0.34 0.45 0.48 0.58
1 1 0-6 1-6 0-15 1-15 1-11
# Ovulation rate measured by real-time ultrasound 30 days after first booster injection given on day 2 of CIRD-PG synchronized cycle Mode of Action of Inhibin Immunization According to our hypothesis, immuno-neutralization of inhibin should lead to a preferential increase in circulating concentration of FSH. In most situations involving native bovine immunogens prepared from follicular fluid and in two independent experiments in which ewes were immunized with recombinant bovine inhibin-tx such an increase in FSH has been confirmed (1, 16, also see 19). In the studies of O'Shea et al. (40) both the degree of inhibin binding and the extent of FSH increase were significantly correlated with the magnitude of ovulation rate increase (r = +0.52, P < 0.001, and r -- +0.39, P < 0.001). There was also a significant correlation between plasma FSH concentration and the coincident specific inhibin binding (r = +0.66, P < 0.001). In the case of sheep immunized against synthetic inhibin-tx peptides variable effects on plasma FSH have been observed (19), despite consistent increases in ovulation rate. This led to suggestions that there may be intra-gonadal effects of inhibin immunization. Recent more intensive blood sampling schedules (3) have shown a transient increase in plasma FSH concentration following primary immunization with peptide 1-32 without any effect on ovulation rate. After a booster immunization an increased ovulation rate was preceded by increased plasma FSH concentration over several days. From this it is inferred that an effective increase in FSH, while not required to be of a large magnitude (24), must be present for some
Theriogenology
9
time to achieve an increase in ovulation rate. Passive immunization of ewes with inhibinbinding antibodies raised against synthetic c~-chainpeptide leads to abrupt increases in plasma FSH concentrations and a three-fold increase in ovulation rate (51). Follicle growth studies using passive immunization (31) support the association between inhibin immunization, plasma FSH concentrations and follicle growth and development. In ewes immunized with peptides mimicking porcine c~-inhibinpeptides (S.T. Anderson, B.M. Bindon, M.A. Hillard and T. O'Shea, unpublished data), antibody binding to l~Ilabelled native 32 kDa inhibin was significantly correlated (n = 76, r = +0.33) with ovulation rate, whereas there was no correlation between ovulation rate and binding to peptide cq-26tracer (n = 76, r = - 0.10). Thus, titres may only be meaningful if measured against the native protein. Heifers immunized with doses of Rec. ovine inhibin-o~.3, varying from 125 I.tg to 1,000 I.tg, show a consistent pattern of inhibin binding (Figure 1), with peak levels evident within seven days of a booster injection. Specific inhibin binding remains elevated for about 14 days, then declines significantly over the next 28 days. Re.boosting promptly induces increased inhibin binding activity. Repeatability of multiple ovulation rate in these inhibin-vaccinated heifers (Table 6) mimics the antibody binding pattern, such that the proportion of cows showing increased ovulation rate declines in the second cycle after booster injection. As in the sheep, plasma FSH concentrations were higher (P<0.05) during the follicular and early luteal phases of the estrous cycle in heifers immunized against Rec. ovine inhibin-c~.3 (Figure 2). In the same study there was no evidence of changes in LH secretion in the immunized females.
Booster 1
Booster 2
40 Level of antigen (Rec. ovine inhibina . 3 /2 ml in vaccine)
30
1000 I,tg 500 I.tg 20 --'-'&
125 I.tg 0 I.tg
10"I
i
250/.tg
i 4
i 8
i 12
i 16
20
W e e k s post-primary immunisation
Figure 1: Inhibin binding in plasma diluted 1:3 in heifers immunized with varying doses of Rec. ovine inhibin-ct.3.
Theriogenology
10
Table 6:
Ovulation rates (OR) in heifers two weeks (synchronised) and five weeks (natural cycle) after boosters 1 and 2 immunization with Rec. ovine inhibin-(z.3 (M. Hillard, B. Bindon, J. Wilkinson, C. Tsonis and J. Findlay, unpublished data) Number with OR >1 Two weeks Five weeks post-booster 1
Treatment group (mg/dose)
Number with OR >1 Two weeks Five weeks post-booster 2
Control
(0)
10
0
0
0
0
I (0.125)
10
5 (2,2,2,3,11)*
1 (2)
4 (2,5,8,1 I)*
1 (3)
2 (0.25
9
5 (2,2,4,8,9)
2 (3,2)
5 (2,3,4,16,37)
2 (2,15)
3 (0.5)
10
6 (2,2,2,3,6,10)
2 (2,2)
5 6 (2,2,2,5,11) (2,2,2,2,8,10)
4 (1.0)
9
4 (2,2,3,3)
2 (2,4)
4 (3,4,5,11)
2 (2,4)
*Individual ORs in brackets.
2.s
.
1"381 i
3
i
|
,
i
21ar0e
i
le y
iati
2G3
•
•
4
to
Figure 2: Plasma FSH before and after prostaglandin (PG) synchronization (10 days postbooster 2) of heifers vaccinated with varying doses (125-1000 gg) of Rec. ovine inhibin-a.3 (see legend Figure 1).
Theriogenology
11
Effects on Puberty Immunization of ewe lambs early in life with inhibins prepared from follicular fluid (2), synthetic inhibin-ct peptides (3, 37) or recombinant bovine-a fusion protein (39) led to some advancement of puberty and increases in ovulation rate (Table 7). It was shown (2, 36) that the effects were more pronounced if the primary immunization was given at age three weeks rather than age nine weeks. In these ewes there were no significant effects of the immunization on plasma FSH or LH concentrations. Table 7:
Effects of immunization of Merino lambs with partially purified bovine inhibin (PPI), recombinant (Rec.ba), synthetic (porcine cq.32) or native (bMPI) inhibin at 3, 7 and 15 weeks of age on inhibin binding (17 weeks) and ovarian activity at 3042 and 72-80 weeks of age (data from 2, 3, 37, 39)
30-42 weeks Immunogen
n
Control PPI
9 9
Control Rec.bcx bMPI
22 25 21
Control Porcine oq.32
12 12
Inhibin binding (%)
Number ovulating
--0.5+0.1 27.2+1.7 12.6+2.3 ---
Ovarian activity 72-80 weeks
OR
Number ovulating
1 6
1.00+0.0 3.33+0.99
9 8
1.00+0.0 2.00+0.57
0 10 3
0 2.95+0.75 2.33+0.88
15 22 15
1.27+0.15 4.41+0.67 1.40+0.16
2 8
1.00+0.0 2.50+0.4
11 11
1.00+0.0 3.10+0.6
OR
In a preliminary study (M.A. Hillard, B.M. Bindon and C.G. Tsonis, unpublished), crossbred beef heifers were immunized with Rec. ovine inhibin-m3 in Montanide:Marcol adjuvant at an average age of 110 days, then boosted 30, 60, 90 and 210 days later. Ovaries , were examined 90 days after the final booster, when the heifers were 14 months old, on average. The data (Table 8) confirm a similar phenomenon to that described in sheep, that is, inhibin vaccination resulted in multiple ovulation with some evidence of advancement of puberty. Table 8:
Treatment
Ovarian activity of beef heifers treated early in life with Rec. ovine inhibin-a.3 and examined at 14 months of age
n
Number with CL or CA
Number of ovulation cycles with OR 1 >1
Control
10
3
4
0
Rec. ovine inhibin-ot.3
11
7
1
10
Ovulation rate (mear~+SE) 1.0+0 3.8+0.5
12
Theriogenology
Inhibin Vaccination and Superovulation for Embryo Transfer Despite the plethora of scientific investigations over the past 15-20 years to refine treatment protocols, variability of superovulation rates and/or numbers of transferable embryos recovered per donor treatment remain as the major limitation to the widespread use of embryo transfer in animal breeding. A reason for this variability is that injection of exogenous gonadotrophins will trigger high peripheral levels of the natural negative feedback regulators (inhibin and estradiol-1713) derived not only from the dominant follicle, but also from the cohort of recruited subordinate follicles which would usually become atretic in an unstimulated estrous cycle (26, 27). The inhibin vaccine approach to superovulation differs in that it is directed at one of these inhibitors that normally counteracts the secretion of endogenous FSH. As a consequence of this approach it is hypothesized that lower dosages of exogenous hormones would be required for superovulation. A dose-response study comparing the ovulatory effects of graded doses of exogenous oFSH(an experimental FSH preparation with 1.8 times the activity of NIH-FSH-S1 and an FSH:LH ratio of 900:1) in control and inhibin-vaccinatedewes (Figure 3) has shown a similar sensitivity of both groups to oFSH treatment (i.e., response slopes parallel), with inhibin vaccination effects on ovulation rate being additive. Thus to attain a set level of superovulation, less exogenous gonadotrophin was required for inhibin-vaccinatedewes.
3-
+
. ~O
O
5..L O
Porcine oq-26
o
Control
O
I
6
b
I
12
Dose of FSH (mg)
Figure 3:
Ovulation rate in control and immunized ewes treated with FSH.
In a preliminary study, Merino ewes were subjected to either a standard PMSG superovulation regime or immunizationwith native bovine inhibin (bMPI 1) or synthetic porcine c~-peptide conjugated to human serum albumin. Embryos were collected surgically and classified by normal embryo assessment standards. The results in Table 9 show significant increases in the number of transferable embryos per synthetic peptide-immunized donor.
Theriogenology Table 9:
13
Ovulation rates and embryo recovery from Merino ewes immunized against inhibins or treated with PMSG (R. Gilchrist, T. O'Shea, G. Hinch and B Bindon, unpublished data)
Immunogen Nil Native bovine Porcine eq.32
n
PMSG
13 11 8
800 iu Nil Nil
Transferable Ovulation Ewes Embryos Percentage embryos rate with per ewe transferable per ewe (meand:SE) embryos (mear~+SE) embryos (mear~+SE) 3.1+0.4 5.0+0.9 11.1+1.9**
10 11 8
1.8+0.4 3.4+0.6 6.4+I .4*
92 65 67
1.7+0.4 2.5+0.6 4.3+1.6*
Porcine eq.32 group significantly different from non-immunized control group ** P < 0.01; * P < 0.05 Preliminary data (23) show that mature Hereford cows immunized against native ovine inhibin (i.e., oMPI with FCA) have increased ovulation rates that resulted in more transferable embryos following mild treatment with exogenous FSH (equivalent to 36 units of NIH oFSH), when compared with non-immunized controls (OR: 16.5+5.6 vs. 2.8+0.5:P<0.05; transferable grade embryos 7.8+2.8 vs. 2.0+0.4: P<0.05). Repeatability of Ovulation Rate in Inhibin-vaccinated Sheep and Cattle In a number of the studies outlined in this review, repeated measures of ovulation rate have been obtained after booster vaccination of ewes or heifers with inhibin antigens. The data have been analysed using least-squares mixed model A N O V A procedures to establish repeatability of ovulation rate. The data in Table 10 show moderate repeatability of ovulation rate in ewes vaccinated with synthetic peptides (particularly tx1.32and tx7.26)and heifers vaccinaTable 10:
Species
Estimates of repeatability of ovulation rates in inhibin-vaccinated sheep and cattle
Antigen
n
OR records per animal
Sheep
Peptide et 1-32 Peptide et 1-26 Peptide ct 7-26 Total e~peptides ~
16 16 15 47
3 3 3 3
Cattle
Rec. o.inhibin.ct.3b oMPIc
16 8
3 or 4 2
Repeatability
P
0.58+0.15 (-)0.07 0.57+0.15 0.41 +0.10
<0.001 NS <0.002 <0.001
0.39+0.16 0.80-2_0.16
<0.003 <0.02
a Post-booster 2 measured through to 10 months (intraclass correlation) b Treated with 500 I.tg fusion protein/vaccination (intraclass correlation) e Superovulatory cycles after booster vacinations 2 and 4, that is, days 87 and 246: Table 3 (repeatability)
14
Theriogenology
ted with 500 I.tg Rec. ovine inhibin-et.3. These estimates are within the range of values (0.40 and 0.69) obtained for prolific Finn sheep and Booroola Merino respectively (42). When the superovulatory cycles of heifers vaccinated with oMPI were analysed, a very high estimate was obtained. This contrasts with the low estimates of repeatability of numbers of ova/embryos (0.23) and transferable embryos (0.15) recovered after superovulation of cattle (21).We know of no comparable data for superovulation in sheep or cattle, despite the perception of industry practitioners that selection of donors with repeatable superovulatory responses is possible. DISCUSSION Knowledge of inhibin biology in ruminants has progressed rapidly since 1985. The availability of pure bovine inhibin- A, synthetic inhibin-ctpeptides and recombinant inhibins has resulted in refinement of early inhibin immunizationstudies and clearer interpretation of the effects observed. Inhibin imrnunogens readily induce increases in ovulation and lambing rates of sheep. The most remarkable effects are: • the increases in ovulation rates after vaccination with quite small peptides (e.g., porcine c~7-26)which persist for more than a year after two booster injections. • the rapid increase in ovulation rate induced by passive immunizationagainst inhibin about the time of luteolysis (51). There is general agreement that vaccination against native and recombinant inhibin causes elevated plasma FSH concentrations in accord with the postulated role of inhibin as a feed-back regulator of FSH synthesis. We have now shown that synthetic inhibin-tx peptide immunogens also act via this pathway. Passive immunization using sera from animals immunized with synthetic peptides (29, 30, 51) confirm that inhibin immunoneutralization induces increased peripheral FSH. Ovulation rate and prolificacy in the cow are more difficult to manipulate by inhibin vaccination. Both native ovine inhibin and recombinant ovine and human inhibins are effective immunogens for inducing increased ovulation rate in the cow. Synthetic peptides have similar effects after multiple booster vaccinations (35). Relatively low levels of 125I-inhibin binding were seen in the cow (i.e., compared to sheep) and some immunogens which result in significant inhibin binding have no effect on ovulation rate. In this review, we provide the first evidence that inhibin immunizationof heifers results in a preferential increase in plasma FSH levels during the follicular phase of the (synchronized) estrous cycle. However, no correlation could be found between the elevated levels of FSH and coincident inhibin binding or subsequent ovulation rate. This may be due principally to the limited (daily) FSI-I concentrations measured. A prototype recombinant ovine inhibin vaccine is currently being evaluated for twinning in cattle. Early fertility and pregnancy results are comparable to previous hormone-based techniques for twinning (4). Recent studies have revealed that the potency of the irnmunogen was far greater than anticipated. Further evaluation may result in an improved technique suitable for commercial application. Inhibin vaccination in the ewe lamb has had variable effects on age at puberty but consistently increased ovulation rate. Early immunizationof the heifer also produces increased ovulation rates with some suggestion of an effect on age at puberty. This aspect requires further research effort. The use of inhibin vaccination holds promise as a means to overcome the variability in superovulation for embryo transfer in sheep and cattle. Preliminary studies outlined in this review reveal that exogenous gonadotrophin treatments can be reduced or eliminated for superovulation in the sheep at least. The recent demonstration (25) that passive inhibin immunoneutralization of cattle on day 12 of the estrous cycle resulted in significant elevation of peripheral FSI-I with coincident increases in the number of small, medium and large ovarian follicles, without the excessive estradiol production observed with exogenous gonadotrophin
Theriogenology
15
treatment (26, 27) supports our contention that inhibin vaccination will improve superovulation regimes. M o d e r a t e to high estimates o f repeatability o f ovulation rates in inhibin vaccinated sheep and heifers also support our hypothesis that less variable repeated superovulation should be achieved with this approach. Future Directions It is essential that any commercial products arising from this technology be acceptable to the general c o m m u n i t y . Further research should be directed at vaccine formulation. In this regard the use o f non-ulcerative aqueous vaccines with novel adjuvants (e.g., s a p o n i n s - Q u i l A, and the i m m u n e s y s t e m cytokines-interleukins and colony stimulating factors) should be investigated. T h e r e is also scope for further investigations o f such aspects as vaccination protocols and i m m u n o g e n dosage using either recombinant or synthetic inhibins. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
AI-Obaidi SAR, Bindon BM, Findlay JK, Hillard MA and O'Shea T. Plasma follicle stimulating hormone in Merino ewes immunized with an inhibin-enriched fraction from bovine follicular fluid. Anim. Reprod. Sci. 1987; 14:39-51. Al-Obaidi SAR, Bindon BM, Hillard MA and O'Shea T. Reproductive characteristics of lambs actively immunized early in life with inhibin-enriched preparations from follicular fluid of cows. J. Reprod. Fertil. 1987; 81:403-414. Anderson ST. 'Inhibin, Peptides and Reproduction in Sheep.' PhD. Thesis, University of New England, Armidale. 1993 Bindon BM and Hillard MA. In: "Twinning in cattle" Cummins LJ. Proc. Aust. Soc. Anim. Prod. 1992; 19:438-447. Bindon BM, Anderson ST, Cummins LJ, Findlay JK, Hillard MA, O'Shea T, Paull D, Tsonis CG and Wilkins JF. Manipulation of reproduction of sheep and cattle by vaccination against inhibin-related peptides. Aust. J. Agric. Res. 1993; in press. Bindon BM, O'Shea T, Miyamoto K, Hillard MA, Piper LR, Nethery RID and Uphill G. Superovulation in pubertal heifers immunized against ovine inhibin purified by monoclonai antibody affinity chromatography. Proc. Aust. Soc. Reprod. Biol. 1988; 20:28 (abstr.). Burger HG. Inhibin: definition and nomenclature, including related substances. J. Endocrinol. 1988; 117:159-160. Crawford RJ, Hammond VE, Evans BA, Coghlan JP, Haralambidis J, Hudson B, Penschow JD, Richards RI and Tregear GW. ¢L-Inhibin gene expression occurs in the ovine adrenal cortex, and is regulated by adrenocorticotropin. Molec. Endocrinol. 1987; 1: 699-706. Cummins LJ, O'Shea T, AI-Obaidi SAR, Bindon BM and Findlay JK. Increase in ovulation rate after immunization of Merino ewes with a fraction of bovine follicular fluid containing inhibin activity. J. Reprod. Fertil. 77:365-372 (1986). Cummins LJ, O'Shea T and Bindon BM. Increased ovulation rates in cattle vaccinated with a partially purified fraction of ovine follicular fluid. Proc. Aust. Soc. Reprod. Biol. 1986; 18:39 (abstr.). Cummins LJ, O'Shea T, Bindon BM, Lee VWK and Findlay JK. Ovarian inhibin content and sensitivity to inhibin in Booroola and control strain Merino ewes. J. Reprod. Fertil. 1983; 67:1-7. Esch FS, Shimasaki S, Cooksey K, Mercado M, Mason AJ, Ying S, Ueno N and Ling N. Complementary Deoxyribonucleic Acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins. Molec. Endocdnol. 1987; 1: 388-396. Findlay JK. An update on the roles of inhibin, activin and follistatin as local regulators of folliculogenesis. Biol. Reprod. 1993; 48:15-23. Findlay JK, Clarke IJ and 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 estrous cycle. Endocrinology 1990; 126: 528-535. Findlay JK, Clarke IJ, Luck MR, Rodgers RJ, Shukovski L, Robertson DM, Klein R, Murray JF, Scaramuzzi RJ, Bindon BM, O'Shea T, Tsonis CG and Forage RG. Peripheral and intragonadal actions of inhibin-related peptides. J. Reprod. Fertil 1991; 43:Suppl. 139-150.
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Theriogenology
16. Findlay JK, Doughton B, Robertson DM and Forage RG. Effects of immunization against recombinant bovine inhibin c~subunit on circulating concentrations of gonadotrophins in ewes. J. Endocrinol. 1989; 120:59-65. 17. Forage RG, Brown RW, Oliver KJ, Attache BT, Devine PL, Hudson GC, Goss WH, Bertram KC, Tolstoshev P, Robertson DM, de Kretser DM, Doughton B, Burger HG and Findlay JK. Immunization against an inhibin subunit produced by recombinant DNA techniques results in increased ovulation rate in sheep. J. Endocrinol. 1987; 114:RI-R4. 18. Forage RG, Ring JM, Brown RW, Mclnerney BV, Corbon GS, Gregson RP, Robertson DM, Morgan FJ, Hearn MTW, Findlay JK, Wettenhall REH, Burger HG and de K.retser DM. Cloning and sequence analysis of eDNA species coding for the two subunits of inhibin from bovine follicular fluid. Proc. Nat. Acad. Sci. USA 1986; 83:3091-3095. 19. Forage RG, Tsonis CG, Brown RW, Hungerford J'W, Greenwood PE and Findlay JK. Inhibin: a novel fecundity vaccine. In "Animal Health and Production for the 21st Century'. (Ed. Beh KJ) CSIRO, Melbourne. pp. 122-130. 1993. 20. Glencross RG, Bleach ECL, McLeod BJ, Beard AJ and Knight PG. Effect of active immunization of heifers against inhibin on plasma FSH concentration, ovarian follicular development and ovulation rate. J. Endocrinol. 1992; 134:11-18. 21. Hahn J. Attempts to explain and reduce variability of superovulation. Theriogenology 1992; 38:269275. 22. Henderson KM, Franchimont P, Lecomte-Yema MJ, Hudson N and Ball K. Increase in ovulation rate after active immunization of sheep with inhibin partially purified from bovine follicular fluid. J. Endocrinol. 1984; 102:305-309. 23. Hillard MA, Bindon BM, King B, O'Shea T, Andrews CM and Hinch GN. Superovulation of cows immunized against native ovine inhibin. Prec. Aust. Soc. Reprod. Biol. 1990; 22:134 (abstr.). 24. Ireland JJ. Control of follicular growth and development. J. Reprod. Fertil. Suppl. 1987; 34:39-54. 25. Kaneko H, Nakanishi Y, Taya K, Kishi H, Watanabe G, Sasamoto S and Hasegawa Y. Evidence that inhibin is an important factor in regulation of FSH secretion during the mid-luteal phase in cows. J. Endocrinol. 1993; 136:35-41. 26. Kaneko H, Terada T, Watanabe G, Taya K and Sasamoto S. Changes in peripheral levels of immunoreactive inhibin and gonadotrophins in cattle induced to superovulate by porcine folliclestimulating hormone. Jpn. J. Anim. Reprod. 1990; 36:77-82. 27. Kaneko H, Watanabe G, Taya K and Sasamoto S. Changes in peripheral levels of bioactive and immunoreactive inhibin, estradiol-1713, progesterone, luteinizing hormone and follicle stimulating hormone associated with follicular development in cows induced to ovulate with equine chorionic gonadotrophin. Biol. Reprod. 1992; 47:76-82. 28. Leversha LJ, Robertson DM, de Vos FL, Morgan FJ, Hearn MTW, Wettenhall REH, Findlay JK, Burger HG and de Kretser DM. Isolation of inhibin from ovine follicular fluid. 1987; J. Endocrinol. 113:213-221. 29. Mann GE, Campbell BK, McNeilly AS and Baird DT. Passively immunizing ewes against inhibin during the luteal phase of the oestrous cycle raises the plasma concentration of FSH. J. Endocrinol. 1989; 123:383-391. 30. Mann GE, Campbell BK, McNeilly AS and Baird DT. The effects of passively immunizing ewes against inhibin and oestradiol during the follicular phase of the oestrous cycle. J. Endocrinol. 1990 125:417-424. 31. Mann GE, Campbell BK, McNeilly AS and Baird DT. Follicular development and ovarian hormone secretion following passive immunization of ewes against inhibin or oestradiol. J. Endocrinol. 1993 136:225-233. 32. Mason AJ, Hayflick JJ, Ling N, Esch F, Ueno N, Ying SY, Guilleman R, Niall H and Seeburg PH. Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-g. Nature 1985; 318:659-663. 33. Meyer RL, Carlson KM, Rivier J and Wheaton JE. Antiserum to an inhibin alpha-chain peptide neutralizes inhibin bioactivity and increases ovulation rate in sheep. J. Anim. Sci. 1991; 69:747-754. 34. Miyamoto K, Hasegawa Y, Fukuda M and Igarashi M. Demonstration of high molecular weight forms of inhibin in bovine follicular fluid (bFF) by using monoclonal antibodies to bFF 32k inhibin. Biochem. Biophys. Res. Commun. 1986; 136:1103-1109. 35. Morris DG, McDermott MG, Diskin MG, Morrison CA, Swift PJ and Sreenan JM. Effect of immunization against synthetic peptide sequences of bovine inhibin ct-subunit on ovulation rate and twincalving rate in heifers. J. Reprod. Fertil. 1993; 97:255-261.
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