Effects of re-immunization of heifers against inhibin on hormonal profiles and ovulation rate

To study the effect of re-immunization against inhibin on ovarian response and hormonal profiles, Japanese beef heifers (n = 5) were re-immunized three times with inhibin vaccine (recombinant ovine inhibin α-subunit in oil emulsion, 125 μg ml−1) one year after the primary immunization. Control heifers (n = 5) were injected with placebo (Montanide: Marcol adjuvant alone). Oestrous cycles were synchronized by using prostaglandin F2α (PGF2α) and ovarian response was monitored daily by ultrasonography. Blood samples were collected by jugular venipuncture for assessment of hormonal levels and inhibin antibody titres. In contrast to controls, inhibin re-immunized heifers generated antibodies against inhibin rapidly reaching a peak level 9 days after the first booster injection. The mean concentrations of FSH in re-immunized cows increased significantly in comparison with controls. In addition, there was a significant increase in oestradiol-17β and progesterone levels in re-immunized cows compared with controls. Inhibin re-immunized heifers had a significant increase in small (≥4 < 7 mm), medium (≥7 < 10 mm) and large (≥10 mm in diameter) sized follicles. Moreover, the mean ovulation rate was 5.0 ± 1.1 after the third booster injection in re-immunized heifers compared with control heifers (single ovulation). These results clearly demonstrate that re-immunization of inhibin can be used to enhance ovarian follicular development and ovulation rate. Furthermore, the great number of follicles is a potential source of oocytes that could be harvested for in vitro fertilization and embryo transfer programmes.

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Effect of active immunization of heifers against inhibin on plasma FSH concentrations, ovarian follicular development and ovulation rate

Journal of Endocrinology ◽

10.1677/joe.0.1340011 ◽

1992 ◽

Vol 134 (1) ◽

pp. 11-18 ◽

Cited By ~ 30

Author(s):

R. G. Glencross ◽

E. C. L. Bleach ◽

B. J. McLeod ◽

A. J. Beard ◽

P. G. Knight

Keyword(s):

Synthetic Peptide ◽

Ovarian Function ◽

Statistical Significance ◽

Follicular Development ◽

Ovarian Response ◽

Active Immunization ◽

Ovulation Rate ◽

Corpora Lutea ◽

Α Subunit ◽

Ovarian Follicular Development

ABSTRACT To study the effects of immunoneutralization of endogenous inhibin on gonadotrophin secretion and ovarian function, prepubertal heifers (n = 6) were actively immunized against a synthetic peptide replica of the N-terminal sequence of bovine inhibin α subunit bIα(1–29)Tyr30) coupled to ovalbumin. In contrast to ovalbumin-immunized controls (n=6), bIα(1–29)Tyr30-immunized heifers had detectable inhibin antibody titres (% binding to 125I-labelled bovine inhibin at 1:2000 dilution of plasma) of 17 ± 3% (s.e.m.) at puberty, rising to 31 ± 5% by the end of the study period 7 months later. Neither age (immunized: 295 ± 8 days; controls: 300 ± 5 days) nor body weight (immunized: 254 ± 13 kg; controls 251 ± 9 kg) at onset of puberty differed between groups. Although the difference did not reach statistical significance, mean plasma FSH concentrations recorded in inhibin-immunized heifers remained 35–40% higher than in controls throughout the 12-week period leading up to puberty (P = 0·14) and during nine successive oestrous cycles studied after puberty (P=0·10). Plasma LH concentrations did not differ between groups at any time during the study. Inhibin immunization had no effect on oestrous cycle length (immunized: 19·8±0·5 days; controls: 19·9±0·5 days). However, in comparison with controls, inhibinimmunized heifers had more medium sized (≥0·5 to <1 cm diameter) follicles during both the preovulatory (95%, P<0·001) and post-ovulatory (110%, P < 0·05 waves of follicular growth and more large (>1 cm diameter) follicles during the preovulatory wave (49%, P<0·05). In addition, the number of corpora lutea observed during the post-ovulatory phase of each cycle was significantly greater in the inhibin-immunized group (43%, P<0·01), as was the recorded incidence of cycles with multiple ovulations (19/56 in the inhibin-immunized group compared with 0/54 in controls; P<0·001). All six inhibinimmunized heifers had at least one cycle with multiple ovulation whereas none of the control heifers did so. These results support the conclusion that immunoneutralization of endogenous inhibin using a synthetic peptide-based vaccine can enhance ovarian follicular development and ovulation rate in heifers. Whether this ovarian response is dependent upon the expected increase in secretion of FSH remains to be established. Journal of Endocrinology (1992) 134, 11–18

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Preovulatory follicle development and luteal function in ewes immunized against a synthetic peptide sequence of the α subunit of bovine inhibin

Journal of Endocrinology ◽

10.1677/joe.0.1330413 ◽

1992 ◽

Vol 133 (3) ◽

pp. 413-419 ◽

Cited By ~ 9

Author(s):

B. J. McLeod ◽

M. G. Hunter ◽

E. C. L. Bleach ◽

R. G. Glencross ◽

J. H. M. Wrathall

Keyword(s):

Synthetic Peptide ◽

Ovulation Rate ◽

Peptide Sequence ◽

Corpora Lutea ◽

Follicle Development ◽

Preovulatory Follicle ◽

Α Subunit ◽

Luteal Function ◽

The Mean

ABSTRACT Immunization against inhibin consistently results in an increase in ovulation rate in sheep, but the effects that this treatment has on follicle development are unknown. In order to determine the influence of inhibin, parameters of follicle development were assessed in ewes that had been actively immunized against a synthetic peptide homologous to the N-terminal sequence (α1–29, Tyr30) of the a subunit of bovine inhibin, a treatment that neutralizes the biological activity of endogenous inhibin. The final stages of preovulatory follicle development that culminate in ovulation were induced in seasonally anoestrous ewes, and follicles were recovered prior to the predicted time of ovulation. After priming with progestagen, inhibin-immunized and control ewes were treated with gonadotrophin-releasing hormone (GnRH) by continuous infusion (200 ng/h). The ovaries were recovered at slaughter 24 h after the start of GnRH treatment and all follicles ≥ 2·0 mm diameter were dissected out and their capacity to produce oestradiol in vitro was assessed. Further groups of similarly treated animals were blood-sampled daily to determine luteal function following GnRH-induced ovulation. The ovaries were recovered from these ewes at slaughter 10 days after the start of GnRH treatment, the corpora lutea were dissected out and their progesterone content was assessed. There were more (P < 0·01) follicles of 5–6 mm diameter (3·2 ± 0·45 (s.e.m.) compared with 1·1 ± 0·25 follicles/ewe) and more (P < 0·001) follicles of > 6 mm diameter (2·8 ± 0·56 compared with 0·9 ± 0·17 follicles/ewe) in inhibin-immunized than in control ewes. In addition, the mean number of the antral follicles that were oestrogenic was greater (P < 0·05) in immunized than in control ewes (2·8 ± 0·66 compared with 1·3 ± 0·25 follicles/ewe). In animals slaughtered 10 days after the start of GnRH treatment, mean ovulation rate was greater (3·17 ± 0·65 and 1·14 ± 0·14, P < 0·01) in inhibin-immunized ewes. Although there was more (P < 0·01) total luteal tissue/ewe in the immunized group, both the mean weight and progesterone content (ng/mg tissue) of individual corpora lutea were similar between treatment groups. Mean plasma progesterone levels increased earlier and reached higher (P < 0·01) mean concentrations in immunized than in control ewes. These results demonstrate that immunization against inhibin increases the number of preovulatory follicles during the follicular phase, and that steroidogenesis within these follicles and the resultant corpora lutea appears to be normal. Journal of Endocrinology (1992) 133, 413–419

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Inhibin immunoneutralization by antibodies raised against synthetic peptide sequences of inhibin α subunit: effects on gonadotrophin concentrations and ovulation rate in sheep

Journal of Endocrinology ◽

10.1677/joe.0.1240167 ◽

1990 ◽

Vol 124 (1) ◽

pp. 167-176 ◽

Cited By ~ 47

Author(s):

J. H. M. Wrathall ◽

B. J. McLeod ◽

R. G. Glencross ◽

A. J. Beard ◽

P. G. Knight

Keyword(s):

Plasma Levels ◽

Synthetic Peptide ◽

Plasma Concentrations ◽

Ovulation Rate ◽

Control Groups ◽

Α Subunit ◽

Blood Samples ◽

Antigenic Properties ◽

N Terminus ◽

The Mean

ABSTRACT Two experiments were conducted to explore the effectiveness of synthetic peptide-based vaccines for active and passive autoimmunization of sheep against inhibin. In the first experiment, adult Romney ewes (n = 20) were actively immunized against a synthetically produced peptide that corresponded to the N-terminus of the α-subunit of bovine inhibin (bIα(1–29)-Tyr30). This peptide was conjugated to tuberculin purified protein derivative (PPD) to increase its antigenic properties. Control groups comprised non-immunized (n = 10) and PPD-immunized (n = 10) ewes. Primary immunization (400 μg conjugate/ewe) was followed by two booster immunizations (200 μg conjugate/ewe), given 5 and 8 weeks later. Following synchronization of oestrus using progestagen sponges, ovulation rates were assessed by laparoscopy. Weekly blood samples were taken throughout the experiment. All inhibin-immunized ewes produced antibodies which bound 125I-labelled bovine inhibin (Mr 32 000), and ovulation rate in inhibin-immunized ewes (2·15 ± 0·22; mean ± s.e.m.) was significantly (P<0·01) greater than in both non-immunized (0·90 ± 0·23) and PPD-immunized (1·20 ± 0·13) control groups. Immunization against the peptide, but not against PPD alone, resulted in a modest rise in plasma FSH, with mean levels after the second boost being significantly (P<0·025) higher (22%) than those before immunization. Moreover, when blood samples were taken (2-h intervals) from randomly selected groups of control (n = 7) and inhibin-immunized (n = 7) ewes for an 84-h period following withdrawal of progestagen sponges, the mean plasma concentration of FSH during the 48 h immediately before the preovulatory LH surge was 37% greater (P< 0·025) in immunized than in control animals. However, more frequent blood sampling (every 15 min for 12 h) during follicular and mid-luteal phases of the oestrous cycle revealed no significant differences between treatment groups in mean plasma concentrations of FSH. In addition, neither mean concentrations of LH nor the frequency and amplitude of LH episodes differed between immunized and control ewes. However, the mean response of LH to a 2 μg bolus of gonadotrophin-releasing hormone, given during the luteal phase, was significantly (P<0·05) less in immunized than in control ewes. These findings indicate that active immunization of Romney ewes against a synthetic fragment of inhibin can promote a controlled increase in ovulation rate, but this response cannot be unequivocally related to an increase in plasma levels of FSH. In the second experiment, passive immunization of seasonally anoestrous ewes (mule × Suffolk crossbred; n = 6 per group) against inhibin, using an antiserum raised in sheep against a synthetic peptide corresponding to the N-terminus of the α-subunit of human inhibin promoted a rapid (<3 h), dose-dependent rise in plasma levels of FSH which remained increased (2·5-fold; P<0·001) for up to 30 h. Plasma concentrations of LH, however, were unaffected by treatment with the antiserum. It is deduced from this observation that, even in the seasonally anoestrous ewe, the ovary secretes physiologically active levels of inhibin, which exert an inhibitory action on the synthesis and secretion of FSH. Journal of Endocrinology (1990) 124, 167–176

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The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology

Reproduction ◽

10.1530/rep.1.00280 ◽

2004 ◽

Vol 128 (4) ◽

pp. 379-386 ◽

Cited By ~ 104

Author(s):

K P McNatty ◽

L G Moore ◽

N L Hudson ◽

L D Quirke ◽

S B Lawrence ◽

...

Keyword(s):

Growth Factors ◽

Point Mutations ◽

Follicular Development ◽

Cumulus Cells ◽

Ovulation Rate ◽

Growth Stages ◽

New Paradigm ◽

Protein Conjugates ◽

Preovulatory Follicles

Ovulation rate in mammals is determined by a complex exchange of hormonal signals between the pituitary gland and the ovary and by a localised exchange of hormones within ovarian follicles between the oocyte and its adjacent somatic cells. From examination of inherited patterns of ovulation rate in sheep, point mutations have been identified in two oocyte-expressed genes, BMP15 (GDF9B) and GDF9. Animals heterozygous for any of these mutations have higher ovulation rates (that is, + 0.8–3) than wild-type contemporaries, whereas those homozygous for each of these mutations are sterile with ovarian follicular development disrupted during the preantral growth stages. Both GDF9 and BMP15 proteins are present in follicular fluid, indicating that they are secreted products. In vitro studies show that granulosa and/or cumulus cells are an important target for both growth factors. Multiple immunisations of sheep with BMP15 or GDF9 peptide protein conjugates show that both growth factors are essential for normal follicular growth and the maturation of preovulatory follicles. Short-term (that is, primary and booster) immunisation with a GDF9 or BMP15 peptide-protein conjugate has been shown to enhance ovulation rate and lamb production. In summary, recent studies of genetic mutations in sheep highlight the importance of oocyte-secreted factors in regulating ovulation rate, and these discoveries may help to explain why some mammals have a predisposition to produce two or more offspring rather than one.

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The conflict between hierarchical ovarian follicular development and superovulation treatment

Reproduction ◽

10.1530/rep-10-0165 ◽

2010 ◽

Vol 140 (2) ◽

pp. 287-294 ◽

Cited By ~ 11

Author(s):

Kenneth P McNatty ◽

Derek A Heath ◽

Norma L Hudson ◽

Karen L Reader ◽

Laurel Quirke ◽

...

Keyword(s):

Granulosa Cell ◽

Granulosa Cells ◽

Follicular Development ◽

Follicular Phase ◽

Ovulation Rate ◽

Cell Populations ◽

Antral Follicles ◽

Functional States ◽

Ovarian Follicular Development

In mammals with a low ovulation rate phenotype, ovarian follicular development is thought to be hierarchical with few, if any, antral follicles at similar stages of development. The hypothesis being tested herein was that if most follicles are in a functionally different state, then the application of exogenous hormones to increase ovulation rate will not overcome the hierarchical nature of follicular development. Using sheep as the experimental model, the functional states of all non-atretic antral follicles ≥2 mm diameter were assessed in individual ewes (N=10/group) during anoestrus with or without pregnant mare's serum gonadotrophin (PMSG) treatment, or after a standard superovulation regimen, or during the follicular phase of the oestrous cycle. The functional states of these follicles were assessed by measuring the FSH- or human chorionic gonadotrophin (hCG)-induced cAMP responses of granulosa cellsin vitro. There were significant overall effects across the treatment groups on the responses of granulosa cells to either FSH or LH (bothP<0.001). It was concluded that for anoestrous ewes with or without PMSG treatment, and ewes during the follicular phase, granulosa cell populations of many follicles (≥2 mm diameter) did not share a similar cAMP response to FSH (∼50% of follicles) or hCG (>90% of follicles) either on a per cell or total cell basis. After superovulation, ≤30 and 10% respectively of the granulosa cell populations shared similar responses to FSH and LH with regard to follicular diameter and cAMP output. Thus, exogenous hormone treatments used routinely for increasing oocyte yield do not effectively override the hierarchical pattern of ovarian follicular development during the follicular phase.

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Induction of lamb follicular development and embryo production in vitro

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236208002428 ◽

2008 ◽

Vol 5 (3) ◽

pp. 257-262 ◽

Cited By ~ 1

Author(s):

Chen Xiao-Yong ◽

Tian Shu-Jun ◽

Sang Run-Zi ◽

Sun Shu-Chun ◽

Zhao Zhu-Jun ◽

...

Keyword(s):

Embryo Transfer ◽

Culture Medium ◽

Ethylenediaminetetraacetic Acid ◽

Follicular Development ◽

Cumulus Cells ◽

Control Group ◽

Embryo Production ◽

The Mean ◽

Development Capability

AbstractEffect of lamb age, transport stimulation and repeated hormone superovulation on the number of collected oocytes were determined. Effect of the culture medium containing ethylenediaminetetraacetic acid (EDTA) on the development of embryos produced in vitro from lamb oocytes was also investigated. Results indicated that the mean numbers of collected and available oocytes (oocytes with compacted cumulus cells, evenly granulated ooplasm and morphologically normal) from 6- to 8-week-old lambs were 60.8±13.9 and 58.2±12.3, respectively. These figures were higher than those obtained from 12- to 14-week-old lambs (27.3±5.1 and 26.0±4.9) (P<0.05). Stimulation by transportation didn't decrease the number of collected oocytes from the superovulated lambs (P>0.05). However, the number of collected oocytes in the repeated superovulation group was significantly reduced, compared with the control group (P<0.05). The embryonic culture medium supplemented with 10 μmol/l EDTA highly improved the development capability of the embryos from lamb oocytes (P<0.05). Healthy lambs were born following embryo transfer.

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Studies on rat ovarian receptors for lutropin (luteinizing hormone). Interaction with β-subunit of sheep lutropin

Biochemical Journal ◽

10.1042/bj1600615 ◽

1976 ◽

Vol 160 (3) ◽

pp. 615-619 ◽

Cited By ~ 9

Author(s):

K Muralidhar ◽

N R Moudgal

Keyword(s):

Biological Activity ◽

Luteinizing Hormone ◽

Cyclic Amp ◽

Chorionic Gonadotropin ◽

Lower Order ◽

Ovarian Response ◽

Β Subunit ◽

Α Subunit ◽

Human Choriogonadotropin

By using radioimmunoassay, the interaction of sheep lutropin (luteinizing hormone, LH) β-subunit with rat ovarian receptors was investigated. The binding of β-subunit was specific, although of much lower order than that of lutropin. Sheep lutropin β-subunit effectively inhibited the binding of human choriogonadotropin (chorionic gonadotropin, gCG) to the ovary, showing that both occupy the same sites. The binding of sheep lutropin β-subunit to ovary was not followed by any detectable increase in cyclic AMP. The ovarian response to lutropin in terms of cyclic AMP production was inhibited in the presence of free β-subunit. The α-subunit of lutropin, when used at concentrations where contamination with whole lutropin was negligible, enhanced the degree of binding of β-subunit; this did not lead to increased cyclic AMP in the tissue. Surprisingly, the binding of β-subunit in vitro was drastically decreased by the prior removal of all endogenous rat lutropin bound to receptors. The implications of these data are discussed in the light of the reported biological activity of the β-subunit.

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Immunisation against inhibin enhances follicular development, oocyte maturation and superovulatory response in water buffaloes

Reproduction Fertility and Development ◽

10.1071/rd10279 ◽

2011 ◽

Vol 23 (6) ◽

pp. 788 ◽

Cited By ~ 25

Author(s):

D. R. Li ◽

G. S. Qin ◽

Y. M. Wei ◽

F. H. Lu ◽

Q. S. Huang ◽

...

Keyword(s):

Oocyte Maturation ◽

Plasma Concentrations ◽

Follicular Development ◽

Oocyte Quality ◽

Control Group ◽

High Group ◽

Α Subunit ◽

And Control

This study was carried out to test the feasibility of enhancing embryo production in vivo and in vitro by immunoneutralisation against inhibin or follistatin. In Experiment 1, multi-parity buffaloes were assigned into three groups: High group (n = 8), which received one primary (2 mg) and two booster (1 mg) vaccinations (28-day intervals) with a recombinant inhibin α subunit in 1 mL of white oil adjuvant; Low group (n = 8), which received half that dose; and Control group (n = 7), which received only adjuvant. Immunisation against inhibin stimulated development of ovarian follicles. Following superovulation and artificial insemination, inhibin-immunised buffaloes had more developing follicles than the Control buffaloes. The average number of embryos and unfertilised ova (4.5 ± 0.6, n = 6) in the High group was higher (P < 0.05) than in the Control group (2.8 ± 0.6, n = 5) and was intermediate (4.1 ± 0.7, n = 7) in the Low group. The pooled number of transferable embryos of the High and Low groups (3.2 ± 0.5, n = 13) was also higher (P < 0.05) than that (1.6 ± 0.7, n = 5) of the controls. The immunised groups also had higher plasma concentrations of activin, oestradiol and progesterone. In Experiment 2, the addition of anti-inhibin or anti-follistatin antibodies into buffalo oocyte IVM maturation medium significantly improved oocyte maturation and cleavage rates following parthenogenic activation. Treatment with anti-follistatin antibody also doubled the blastocyst yield from activated embryos. These results demonstrated that immunisation against inhibin stimulated follicular development, enhanced oocyte quality and maturation competence, yielded more and better embryos both in vivo and in vitro.

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Increased production of sheep embryos following superovulation of Merino ewes with a combination of pregnant mare serum gonadotrophin and follicle stimulating hormone

Reproduction Fertility and Development ◽

10.1071/rd9910551 ◽

1991 ◽

Vol 3 (5) ◽

pp. 551 ◽

Cited By ~ 42

Author(s):

JP Ryan ◽

JR Hunton ◽

WM Maxwell

Keyword(s):

Follicle Stimulating Hormone ◽

Ovarian Response ◽

Ovulation Rate ◽

Corpora Lutea ◽

Embryo Recovery ◽

Designed Experiment ◽

The Mean ◽

Mean Concentration ◽

Pregnant Mare Serum Gonadotrophin ◽

Stimulating Hormone

In a factorially designed experiment (N = 321), 0, 800 or 1600 I.U. pregnant mare serum gonadotrophin (PMSG) were administered in combination with 0, 12 or 18 mg follicle stimulating hormone (FSH-P) to superovulate Merino ewes in autumn and spring. A moderate dose of PMSG (800 I.U.) in conjunction with 12 or 18 mg FSH-P increased the ovulation rate above that observed when FSH-P was used alone. This was accomplished by (i) increasing the proportion of ewes that exhibited a superovulatory response (greater than 3 corpora lutea (CL) or persistent large follicles (LF): 69/70 (99%) v. 55/74 (74%), P less than 0.001), and (ii) in those ewes that exhibited a superovulatory response, by an additive effect of exogenous gonadotrophin (14.8 +/- 0.9 CL (69) v. 11.3 +/- 0.9 CL (55), P less than 0.01) without increasing the incidence of LF. The use of 1600 I.U. PMSG in conjunction with 12 or 18 mg FSH-P was characterized by an increase in the number of LF and, in comparison with 800 I.U. PMSG, a reduction in ovulation rate. Season had no effect on the numbers of CL, but total ovarian response (CL + LF) was higher in autumn than in spring (P less than 0.01), because of a greater incidence of LF (P less than 0.001). The proportion of ewes with regressed CL was higher in autumn than in spring (53/143 (37%) v. 32/156 (21%), P less than 0.01), and increased with increased dose of gonadotrophin. Furthermore, a nutritional component to the incidence of ewes with regressed CL was suggested by the observation that the mean concentration of plasma glucose was higher for ewes with normal CL than for ewes with regressed CL (P less than 0.05). Rates of ova or embryo recovery, fertilization and embryo development generally declined with an increase in the incidence of LF as a result of increases in the dose of gonadotrophin and season of administration.

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