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 alpha-subunit in oil emulsion, 125 microg 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 F2alpha (PGF2alpha) 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-17beta and progesterone levels in re-immunized cows compared with controls. Inhibin re-immunized heifers had a significant increase in small (> or =4 < 7 mm), medium (> or =7 < 10 mm) and large (> or =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.

Effect of transcutaneous immunization with co-administered antigen and cholera toxin on systemic and mucosal antibody responses in sheep.

Direct application of antigens to skin together with an adjuvant, a procedure called transcutaneous immunization (TCI), can induce systemic immune responses in mice, humans, cats and dogs. In previous studies we found that cholera toxin (CT) applied topically on unbroken skin induces systemic antibody and lymphocyte proliferative responses in sheep. The current study examined whether concurrent administration of CT and tetanus toxoid (TT) delivered transcutaneously could induce specific antibody responses to both antigens in sheep. Antibodies to both TT and CT were induced by TCI although antibody titres in serum to TT were higher in sheep receiving TT plus alum by intramuscular injection (n=5) than TT plus CT by TCI (n=5). The ratio of IgG1/IgG2 antibody to TT in serum was near unity, and the route of immunization, TCI versus injection, did not influence this ratio. In contrast, the ratio of IgG1/IgG2 antibody differed significantly between the two antigens, TT and CT, delivered by TCI, with a higher proportion of IgG1 antibody in serum to CT than TT. Antibody to TT was detected in lung washes from TCI and injection groups, with IgG1 predominating over IgG2 in both groups. IgA antibodies to CT and TT were detected in sera of CT and TT-immunized groups respectively but in lung washes IgA antibody to TT was detected only in the injection group. Results show that TCI induced systemic antibody responses to CT and the co-administered antigen TT, whereas no evidence was obtained for mucosal IgA responses following TCI.

Development of embryos in superovulated guinea pigs following active immunization against the inhibin alpha-subunit.

Embryo recovery and subsequent embryonic development from guinea pigs treated with or without inhibin vaccines were compared to determine the effect of active immunization against the inhibin alpha-subunit. Twenty female guinea pigs of the Hartley strain were injected 3 times either with 1 ml inhibin vaccine (recombinant ovine inhibin a-subunit in oil emulsion: 50 microg/ml, inhibin-immunized group), or 1 ml placebo (saline in oil emulsion; control group) at 4 week intervals. After one estrous cycle following the last injection, females were naturally mated and embryos were collected at 11:00 hr of day 6 of pregnancy (Day 1: sperm in the vaginal smear) for culture in vitro. Active immunization increased the number of corpora lutea (12.6+/-3.0 vs. 4.6+/-0.2, P<0.05), recovered embryos (9.8+/-1.9 vs. 3.6+/-0.4, P<0.01) and normal embryos (7.8+/-1.4 vs. 3.6+/-0.4, P<0.05), although estrous cycle length was not affected (P>0.05). During subsequent 8 day culture in vitro, most of the recovered embryos formed trophoblast outgrowth; 100% (14/14) and 88.2% (15/17) in control and immunized groups, respectively. High levels of inhibin antibody titers were sustained in the inhibin-immunized guinea pigs at least for 5 months after the last injection while no antibody titer was detected in the control animals. These results indicate that active immunization against the inhibin a-subunit is a long-acting and efficient method to induce superovulation with normal embryonic development in the guinea pig.

Follicle selection in cyclic guinea pigs with active immunization against inhibin alpha-subunit.

Experiments were conducted to elucidate the mechanisms of active immunization against inhibin on ovarian follicular development and selection in guinea pigs. Estrous cycle was synchronized in experimental guinea pigs by implanting progesterone containing tubes. Antibodies that bound 125I-labeled bovine inhibin were produced by all guinea pigs receiving the inhibin vaccine (recombinant ovine alpha-subunit in oil emulsion) without any effects on duration of the estrous cycle. Active immunization against inhibin increased the plasma concentrations of progesterone during the luteal phase and the plasma concentrations of estradiol but failed to increase the plasma concentration of follicle-stimulating hormone (FSH) during preovulatory period. The treatment also increased the number of corpora lutea (from 1.3+/-0.3 to 7.0+/-1.6 per each ovary), and preovulatory sized follicles (from 1.8+/-0.6 to 7.0+/-1.6 per each ovary), and follicles stained positively for inhibin alpha-subunit (from 2.3+/-0.5 to 6.3+/-1.3 per each ovary) significantly. The results indicate that active immunization against inhibin enhances ovulation rate by affecting the follicle selection and only dominant follicle can be stained for inhibin alpha-subunit in guinea pigs. This study is firstly to provide direct evidence that inhibins play important role in follicle selections in guinea pigs.

Induction of superovulation by inhibin vaccine in cyclic guinea-pigs.

Experiments were conducted to determine whether neutralizing endogenous inhibin affects follicular development and ovulation rate in guinea-pigs. Eighteen female guinea-pigs bearing 4 week progesterone implants were divided into three groups. At 1 week after removal of the progesterone implants, the animals were given a s.c. injection of 1 ml placebo (saline in oil emulsion; control), or 25 or 50 micrograms inhibin vaccine three times at 4 week intervals. Blood samples were collected once a week throughout the experiment for measuring inhibin antibody titres. After the third injection of inhibin vaccine, blood samples and ovaries were collected on the morning of day 8 after the day of oestrus. Inhibin vaccine increased the ovulation rate in a dose-dependent manner (placebo: 4.2 +/- 0.4; 25 micrograms inhibin vaccine: 6.2 +/- 0.9; 50 micrograms inhibin vaccine: 9.8 +/- 0.9) without any effects on the duration of the oestrous cycle. The results also showed that active immunization against inhibin increased the number of atretic follicles of 300-399 microns in diameter on day 8 after ovulation. The present study is the first to show that the active immunization against inhibin may be a useful method for inducing multiple ovulation in guinea-pigs.

Induction of systemic immune responses in sheep by topical application of cholera toxin to skin.

Cholera (and related) toxins (CT) when applied topically on unbroken skin induce systemic immune responses in mice, a procedure called transcutaneous immunization (TCI). The current study examined the capacity for TCI to induce systemic immune responses in sheep. Three groups (n=5 per group) were immunized at day 0 (priming) and day 28 (boosting) with 250 microg of CT in water by TCI, with 25 microg of CT in alum by intramuscular injection, or not immunized. Serum samples were taken at days 0, 28, 42, 56 and 70 after immunization for measurement of CT-specific IgG as well as CT-specific IgG1, IgG2, IgA and IgM antibodies by ELISA. After immunization, IgG, IgG1 and IgG2 antibody in immunized groups were significantly higher than in the control group, and boosting further increased these titres. IgG, IgG1 and IgG2 in the injection group were significantly higher than in the TCI group. There was a preponderance of IgG1 antibody, relative to IgG2, in both immunized groups. CT-specific IgA and IgM were detected in both immunized groups. Lymphocyte proliferation to CT was measured at day 90. A CT-specific lymphocyte proliferative response (stimulation index>2) was detected in all sheep from the injection group, in two sheep from the TCI group and in none of the controls. Results demonstrated that TCI induces primary and secondary antibody responses and specific proliferative responses to CT in sheep.

The effect of acute immuno-neutralisation of inhibin in ewes during the early luteal phase of the oestrous cycle on ovarian hormone secretion and follicular development.

Ewes with ovarian autotransplants received either inhibin antiserum (10 ml i.v. raised in sheep against recombinant 32 kDa human inhibin; n = 6) or sheep serum (10 ml i.v.; n = 5) on day 3 of the luteal phase with additional daily injections (1 ml i.v.) from 48 h after the initial bolus until day 13. Jugular and ovarian venous blood samples were taken 4-hourly over days 2-13 of the luteal phase. Blood samples were also taken at more frequent intervals (every 10-15 min for 2-3 h) to examine pulsatile secretory responses from the ovary to endogenous and gonadotrophin-releasing hormone-induced (150 ng i.m.) LH pulses on days 4, 6, 8, 10 and 12 of the luteal phase. Plasma FSH levels, ovarian steroid secretion and ovarian follicular development were measured. The ovarian follicle population was estimated daily by real time ultrasound scanning. Immunisation against inhibin resulted in a 3- to 4-fold increase (P < 0.001) in plasma FSH levels within 8 h with levels remaining elevated over controls for 6-7 days. Within 24 h of immunisation there was an increase in the number of small ovarian follicles (P < 0.05) and by 3 days after treatment immunised ewes had 4-6 large ovarian follicles/ewe with this increase in the total number of large follicles being maintained for the rest of the experimental period (P < 0.05). Mean ovarian oestradiol secretion during intensive bleeds was not different from controls 24 h after immunisation, but by 3 days after immunisation it was elevated 4- to 5-fold (P < 0.001) over controls with this increase being maintained throughout the experiment. Similar responses to immunisation against inhibin in androstenedione secretion were observed although mean androstenedione secretion was not elevated until 7 days after treatment. In vitro antibody titres in immunised ewes remained elevated but declined steadily (P < 0.001) over the experimental period. We conclude that the initial stimulation of follicle development and ovarian steroid secretion following passive immunisation against inhibin can be attributed to increased blood FSH. However, the fact that with time FSH declined but increased follicle development was sustained, despite maintenance of high circulating antibody titres, suggests that on a longer term basis inhibin immunisation may stimulate ovarian function by interfering with the modulation of follicle development by inhibin at an ovarian level.

Immunological manipulation of ovulation rate for twinning in cattle.

Unlike in sheep, in which immunization against androstenedione causes mild and reasonably controlled increased ovulation rate, in similar studies cattle showed highly variable responses ranging from increased ovulation rate and fertility through to anovulation/anoestrous or superovulation. As a consequence, interest in manipulation of ovulation rate through this approach has declined and is now focused on immunological manipulation of endogenous inhibin following successful studies in sheep. Studies have concentrated on developing a prototype inhibin-based vaccine to be used for twinning in the Australian beef industry. The prototype vaccine (with recombinant ovine inhibin-alpha.3 fusion protein and Montanide:Marcol adjuvant) has proved to be very potent and control of the degree of ovarian stimulation has not been possible. The proportion of cattle with increased ovulation rate after inhibin immunization is affected by timing of booster vaccination within the ovarian cycle, time after vaccination, vaccine formulation and possibly genotype. Physiological studies show that cattle responding to the inhibin vaccine have increased plasma inhibin binding of native bovine inhibin, high plasma FSH concentrations, greater numbers of large (> or = 8 mm) follicles and fewer small (< 5 mm) follicles during the preovulatory wave of follicular development compared with control or non-responding animals. Significant correlations among the response parameters (i.e. inhibin binding, plasma FSH concentrations, number of large follicles and ovulation rate) have been demonstrated. The results indicate that greater understanding of the various processes of folliculogenesis will be necessary to achieve a controlled increase in ovulation rate in cattle.

Manipulation of inhibin during the luteal-follicular phase transition of the primate menstrual cycle fails to affect FSH secretion.

The pattern of inhibin concentrations in blood during the menstrual cycle in primates has suggested an endocrine role of inhibin in the negative feedback control of FSH secretion during the luteal phase. Conversely, the fall in inhibin during the late luteal phase may play a role in the rise in serum FSH during the luteal-follicular phase transition. This hypothesis was examined by determining the effects of manipulation of inhibin on FSH secretion in stumptailed macaques. During the mid-luteal phase the putative inhibin feedback was inhibited by i.v. administration of 20 ml of ovine antiserum to human recombinant inhibin in 4 macaques. FSH secretion was unaffected during the initial 24 h period post-treatment and the timing of the rise in FSH which occurred during the subsequent luteal-follicular phase transition was normal. To determine whether the elevated serum concentrations of FSH observed during the early follicular phase could be reduced by administration of inhibin, 5 cyclic macaques were treated with 200 micrograms of recombinant human inhibin i.v. Serum FSH concentrations were unaltered. These results suggest that inhibin does not play a major role in modulating FSH secretion during the luteal-follicular phase transition.

Effect of active immunization against the amino-terminal peptide (alpha N) of the alpha 43 kDa subunit of inhibin (alpha 43) on fertility of ewes.

Immunization against the amino-terminal peptide (alpha N) of the alpha 43 subunit of inhibin was shown previously to reduce fertility in ewes. The aim of this study was to examine the effects of active immunization of ewes against alpha N on egg recovery and fertilization rates. Ewes were immunized against alpha N immunogen, and were given 800 I.U. of pregnant mare's serum gonadotrophin at the end of treatment with intravaginal progesterone to synchronize the oestrous cycles. Control ewes received adjuvant only. The ewes were run with fertile rams, and 4 days after withdrawal of the progesterone device the oviducts were flushed to recover eggs and luteal structures on the ovaries were recorded. Eggs were recovered from 17/19 (90%) control ewes compared with 4/16 treated ewes (25%) (P < 0.01), and the egg recovery rates were 76% (45/59) and 17% (7/42) respectively (P < 0.001). The mean number of corpora lutea (CL) per ewe were similar (3.1 +/- 1.4 v. 2.6 +/- 1.0) but several CL in the treated ewes did not appear to have ruptured, and 2 treated ewes had cystic follicles and no CL. There were no apparent differences in either the fertilization rates or the stages of development of fertilized eggs between treated and control ewes. Antibody binding levels in follicular fluid were approximately half those found in peripheral plasma. It is concluded that immunization of ewes against alpha N leads to lowered fertility by suppressing ovulation, implicating alpha N in the normal ovulatory process.

Pituitary and ovarian function in ewes immunized against the amino-terminal peptide (alpha N) of the inhibin alpha 43-subunit.

Immunization of ewes against the amino-terminal peptide (alpha N) of the pro-alpha-subunit of inhibin has been shown to reduce fertility, thought to be due to disruption of ovulation. The aims of this study were to examine the effects of active immunization of ewes against alpha N on circulating concentrations of FSH, LH and on ovarian inhibin and progesterone, and to relate these observations to number of corpora lutea and oocyte recovery rates. Ewes were immunized against one or both of two recombinant full length bovine-alpha N immunogens (FP1 and FP2). Three experiments were performed in which jugular venous plasma was sampled from control and immunized ewes: (1) hourly across the oestrous surge of gonadotrophins (Expt 1); (2) daily for one entire oestrous cycle, and in the subsequent cycle, oviducts were flushed to recover ovulated eggs (Expt 2); and (3) samples were taken at 10 min intervals during the follicular and luteal phases (Expt 3). Binding of 125I-labelled alpha N1-26 to serum was greater (P < 0.05) in immunized groups than in controls for all experiments. The number of eggs per corpus luteum recovered from the oviducts was lower (P < 0.05) in the alpha N-immunized groups (39%) than in controls (88%). There were more (P < 0.05) corpora lutea per ewe in FP2 immunized groups 4 (1.8 +/- 0.45) and 5 (1.75 +/- 0.5) than in the control group (1.13 +/- 0.13), but no increase in group 3 (FP1; 1.4 +/- 0.24).(ABSTRACT TRUNCATED AT 250 WORDS)

Active immunization of Merino ewe lambs with recombinant bovine alpha inhibin advances puberty and increases ovulation rate.

Ewe lambs (n = 24-25) were immunized at 3, 7 and 15 weeks of age with recombinant bovine alpha-inhibin (rec inhibin) or with bovine monoclonal antibody purified inhibin (bMPI) obtained by immunochromatography from bovine follicular fluid or with adjuvant alone (control). Antibodies in the plasma of the lambs immunized with the inhibin preparations bound to iodinated 31 kDa bovine inhibin. Binding was minimal after the primary immunization, increased after each booster immunization and remained elevated until at least 45 weeks of age (29% for rec inhibin and 11% for bMPI). Of the group treated with rec inhibin, 10 ovulated as lambs (control 0/22; bMPI 3/21) and onset of overt oestrous activity (as hoggets) was advanced (P < 0.05) by 17 days in lambs immunized with rec inhibin. As hoggets, the ovulation rate was greater (P < 0.01) in the rec inhibin immunized lambs (4.41 +/- 0.67) than in the control animals (1.27 +/- 0.15) but not in the bMPI-treated lambs (1.40 +/- 0.16). After a further immunization at 17 months of age, however, ovulation rate increased (P < 0.01) in the bMPI-immunized group (3.40 +/- 0.47) but was unchanged in the lambs immunized with rec inhibin (2.80 +/- 0.52) and those in the control group (1.15 +/- 0.08). There were no effects of immunization on plasma concentrations of either follicle stimulating hormone (FSH) or luteinizing hormone (LH). The initial smaller response seen with the bMPI may be due to either the presence of compounds other than inhibin in such preparations or the small absolute amount of inhibin injected.(ABSTRACT TRUNCATED AT 250 WORDS)

The antagonistic effect of exogenous LH pulses on FSH-stimulated preovulatory follicle growth in ewes chronically treated with a gonadotrophin-releasing hormone agonist.

The hypogonadotrophism model induced by the chronic administration of gonadotrophin-releasing hormone (GnRH) agonist was used to investigate the effects of different concentrations of FSH with or without LH pulses on the stimulation of follicular development in the ewe. Continuous administration of an agonist (buserelin) by osmotic minipump to thirty-six Welsh Mountain ewes from the early luteal phase for 5 weeks resulted in a sustained suppression of the plasma concentration of FSH and inhibited the pulsatile release of LH. The inhibition of gonadotrophin secretion was due to the desensitization and/or down-regulation of pituitary gonadotroph function, since the agonist-treated animals showed no response to a challenge of 1 microgram GnRH. During week 6 of agonist treatment, ewes were infused with either 4-hourly pulses of ovine LH (9 micrograms/pulse), low concentrations of ovine FSH (3 micrograms/h) or high concentrations of FSH (9 micrograms/h) alone or with 4-hourly pulses of LH. After 5 days of gonadotrophin infusion, there was no difference between the mean number of follicles per ewe from the animals treated with LH alone, low concentrations of FSH with or without LH pulses or the high concentration of FSH alone compared with the mean number of follicles from control ewes on day 8 of the luteal phase. Infusion of the high concentration of FSH alone stimulated the development of an increased number of large oestrogenic follicles (follicles greater than 2.5 mm in diameter and secreting greater than 3.7 nmol oestradiol/h in vitro) compared with control ewes. The addition of high-amplitude LH pulses to the infusion of the high concentration of FSH prevented follicles developing beyond 2.5 mm in diameter, but doubled the number of small follicles (less than or equal to 2.5 mm) present in the ovaries. These results show that normal follicular development can be induced by physiological concentrations of FSH alone in the absence of pulsatile LH release. The addition of high-amplitude LH pulses antagonized this stimulatory effect of FSH on follicle growth in the ewe.

Immunization against recombinant bovine inhibin alpha subunit causes increased ovulation rates in gilts.

Immunization of gilts in a commercial piggery against a fusion protein of the alpha subunit of bovine inhibin, produced by recombinant DNA methods, resulted in mean ovulation rate increases of 35% at the oestrus at which, under the piggery's management practices, they would have been mated. Sera from two immunized groups showed mean binding of 6.6% and 4.9% when assayed, at 1:800 final dilution, against iodinated bovine inhibin (Mr 31,000). Ovulation rates of immunized gilts were highly correlated with the ability of serum to bind iodinated native inhibin (r = 0.62; P less than 0.001), particularly when weight and age were included in the correlation (r = 0.72; P = 0.001), and inhibin binding accounted for 38% of the total variation in ovulation rate. Immunization caused no deleterious effects on growth rate or onset of oestrus. These results demonstrate the potential for use of such immunization to increase prolificacy in gilts and young sows.

FSH causes a time-dependent stimulation of preovulatory follicle growth in the absence of pulsatile LH secretion in ewes chronically treated with gonadotrophin-releasing hormone agonist.

The study investigated the relationship between the plasma concentration of FSH and the stimulation of preovulatory follicle growth in vivo in ewes chronically treated with the gonadotrophin-releasing hormone (GnRH) agonist buserelin (HOE 766). Welsh Mountain ewes with regular oestrous cycles were treated for 6 weeks with two discs implants placed s.c., each containing 5 mg of the agonist in a matrix of polyhydroxybutyric acid. Treatment with the agonist for 35 days produced a sustained suppression of the plasma concentration of FSH, stopped the pulsatile release of LH and prevented follicular development beyond 2.5 mm diameter. There was no difference between the total number of follicles greater than 1.0 mm diameter present in the ovaries of GnRH agonist-treated ewes and day 8 luteal phase control ewes. During the sixth week of agonist treatment ewes were infused with ovine FSH (6 micrograms NIADDK-oFSH-16/h) in the presence of only basal concentrations of LH. After 24, 48, 72 or 120 h of FSH infusion, the mean number of follicles greater than 1.0 mm diameters per ewe was not significantly different between treated and control animals. Infusion of FSH caused a time-dependent increase in (1) the number of follicles per ovary greater than 2.5 mm, (2) the mean diameter of these follicles and (3) the proportion of the large follicles which could be classified as oestrogenic (greater than 3.7 nmol oestradiol/follicle per h in vitro). Injection of human chorionic gonadotrophin (750 IU i.m.) after 120 h of FSH infusion caused the majority of these large follicles to ovulate and form apparently normal corpora lutea. These results indicate that, in the absence of pulsatile LH, FSH stimulates the growth of normal large oestrogenic follicles which, when stimulated, ovulate to produce viable corpora lutea.

Immunisation against the amino-terminal peptide (alpha N) of the alpha 43 subunit of inhibin impairs fertility in sheep.

Processing of the 58 kDa to the 31 kDa form of inhibin (Inh) involves cleavage of the amino-terminal peptide (alpha N) from the alpha 43-subunit. We show that active immunisation of female sheep against a recombinant bovine alpha N impairs their fertility. In Exp 1, 5 treated (Group 1; 300 micrograms alpha N) and 6 control ewes (Group 2; adjuvant only) were immunized (Day 1) and given boosters on Days 22 and 56. In Group 1, mean +/- SEM binding of 125I-31 kDa Inh was less than 0.5% on Days 33 and 44, whereas binding of 125I-58 kDa Inh was 4.9 +/- 0.7 and 6.2 +/- 0.6%, respectively. In Group 2 binding of both tracers was less than 0.5%. The corpora lutea (CL)/ewe in Group 1 on Days 44 and 82 were 1.8 +/- 0.2 and 2.8 +/- 0.9, respectively, and were not different from those in Group 2 (1.7 +/- 0.3 and 1.5 +/- 0.2, respectively). One ewe in Group 1 versus 5/6 ewes in Group 2 were diagnosed pregnant. In Exp 2, 18 treated and 16 controls were immunized as in Exp 1. The binding of 125I-58 kDa Inh in treated ewes (2.4 +/- 0.3%) was greater than in controls (less than 0.5%) on Day 56. The CL/ewe in treated ewes (1.8 +/- 0.2) was similar to that in controls (2.0 +/- 0.1) on Day 76. All 16 control ewes but only 7/17 treated ewes were subsequently diagnosed pregnant. The plasma progesterone concentrations were similar in treated ewes which did (7.6 +/- 1.2 nmol/L) and did not (7.0 +/- 0.7) become pregnant. Neither basal nor GnRH-stimulated concentrations of LH, nor basal concentrations of Inh differed between treated and controls in Exp 2. Similarly, there were no differences in FSH, except that basal concentrations were higher in the luteal phase of treated ewes. We conclude that immunisation of ewes against alpha N results in a significant reduction in fertility.

Effect of level of food intake of ewes on the secretion of LH and FSH and on the pituitary response to gonadotrophin-releasing hormone in ovariectomized ewes.

The effect of level of food intake on LH and FSH profiles and pituitary sensitivity to gonadotrophin-releasing hormone (GnRH) was investigated in two groups of 12 ovariectomized ewes. Ewes with a high intake (group H) had a mean daily intake (+/- S.E.M.) of 1.99 +/- 0.075 kg dry matter (DM)/head per day while ewes with a moderate intake (group M) consumed a mean of 1.02 +/- 0.021 kg DM/head per day. Ovaries were surgically removed from six ewes of each group on day 11 of the luteal phase and from the remainder 30 h after an injection of 100 micrograms prostaglandin analogue given on day 11 to induce luteolysis. During both the luteal phase and the follicular phase, mean LH and FSH concentrations and LH pulse frequencies and amplitudes were unaffected by the level of intake but mean plasma prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes in the follicular phase. Mean LH and FSH concentrations at day 2 after ovariectomy were unaffected by treatment while mean prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes. At day 7 after ovariectomy, mean LH and FSH concentrations were lower (P less than 0.05) in group H than in group M ewes although mean LH pulse frequencies and pulse amplitudes were not significantly affected by the level of intake at either time.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a major role of inhibin in the feedback control of FSH in the male rat.

Adult rats (16-18/group) received a single intratesticular injection of 25, 100 or 400 microliters glycerol solution (7:3 in distilled water, v/v). Half of the rats in each group were given implants of testosterone, a testosterone-filled Silastic capsule (1.5 cm length) to provide serum values of testosterone within the normal range. After 1 week all animals were killed by decapitation. Serum concentrations of gonadotrophins, testosterone and immunoactive inhibin as well as testicular concentrations of testosterone and bioactive inhibin were determined. Testicular histology was studied in Paraplast-embedded tissue stained with PAS and haematoxylin-eosin. Glycerol treatment caused a dose-dependent ablation of spermatogenesis in a distinct area around the site of injection. Serum concentrations of FSH increased proportionally with increasing spermatogenic damage while serum LH and testosterone remained unaltered except with the highest glycerol dose. The rise in serum FSH was significantly correlated with serum (r = -0.70, P less than 0.001) and testicular (r = -0.66, P less than 0.001) concentrations of inhibin. A less pronounced correlation was found between LH and serum inhibin (r = 0.48). No correlation was found between the concentrations of LH and testicular inhibin or between serum concentrations of FSH and serum testosterone in the 25 and 100 microliters groups. Maintenance of low to normal serum testosterone concentrations by means of Silastic implants blocked the elevation of FSH in glycerol-treated animals but failed to affect significantly serum FSH in untreated rats. In all testosterone treated rats testicular inhibin concentrations were markedly reduced in the presence of lowered concentrations (7-14%) of testicular testosterone and unaltered serum FSH concentrations.

Changes in the plasma concentrations of inhibin throughout the normal sheep oestrous cycle and after the infusion of FSH.

A radioimmunoassay for inhibin was developed using a peptide containing the 1-26 amino acid sequence of the N-terminus of the alpha-chain of 32 kDa porcine inhibin as immunogen, and 125I-labelled tracer. Evaluation of this assay using Sephadex column chromatography, chromatoelectrophoresis and immunoblotting confirmed that it measured all forms of inhibin present in sheep follicular fluid and was suitable for measurement of inhibin in sheep plasma. There was no evidence of the presence of free alpha-subunit in either sheep follicular fluid or ovarian vein plasma. The concentration of inhibin in jugular plasma throughout the follicular and luteal phases of four ewes with ovarian autotransplants was measured. The ovarian secretion of inhibin and oestradiol were also measured simultaneously throughout the follicular phase in a spontaneous cycle and after infusion of NIH-oFSH-S14 at 10 micrograms/h for 48 h following premature luteal regression induced by prostaglandin. The results showed: (1) no change in the peripheral concentration of inhibin throughout the cycle except an increase related to the periovulatory increase in FSH and LH. (2) Following luteal regression, the concentration of FSH fell as the secretion rate of oestradiol increased. During this time there was no significant change in the peripheral concentration of inhibin or ovarian inhibin secretion rate. (3) Following the infusion of FSH there was a marked increase in the concentration of inhibin in both ovarian and peripheral plasma and an increase in ovarian inhibin secretion rate. (4) The calculated metabolic clearance rate of inhibin, 20.3 ml/min, is similar to that of FSH. We conclude that in the ewe the ovarian inhibin secretion rate is stimulated by FSH and, although inhibin may modulate the basal secretion of FSH, a change in its secretion does not account for the fall in FSH which occurs during the follicular phase of the sheep oestrous cycle.

Factors affecting the secretion of immunoactive inhibin into testicular interstitial fluid in rats.

Immunoreactive inhibin was measured in testicular interstitial fluid (IF) from rats during sexual maturation or after impairment of spermatogenesis induced by ethane dimethanesulphonate (EDS), unilateral cryptorchidism or local heating (43 degrees C, 30 min) of the testes, to ascertain its usefulness as a marker of changing Sertoli cell function. Cultures of isolated seminiferous tubules were also studied. Inhibin was measured by a radioimmunoassay directed towards the first 26 amino acids of the N-terminus of the alpha-subunit, and the results confirmed for selected pools of IF by in-vitro bioassay using dispersed ovine pituitary cells. During puberty, IF levels of immunoactive inhibin fell by more than 90% (P less than 0.001) between 30 and 60 days of age, a decrease paralleled by the levels of androgen-binding protein (ABP), another Sertoli cell product secreted into IF. These changes also paralleled, but preceded, the fall (60%; P less than 0.001) in serum levels of FSH between 40 and 70 days, while the serum and IF levels of testosterone increased more than two-fold over this period. When adult rats were injected with EDS to destroy the Leydig cells, testosterone levels in IF and serum were undetectable at 3 and 7 days after treatment, were just detectable at 14 days and thereafter returned slowly towards normal by 42 days. The initial androgen withdrawal following EDS treatment caused a progressive reduction in testicular weight up to 21 days and this was accompanied by a significant increase in the serum levels of FSH and a two- to threefold increase in the IF levels of immunoactive inhibin (and also of ABP). Serum FSH and IF levels of immunoactive inhibin returned to within the normal range by 42 days when testosterone levels had normalized. In contrast, in two other experimental situations in which a marked decrease in testicular weight coupled with an increase in IF levels of ABP occurs, different results for the IF levels of immunoactive inhibin were obtained. Thus, in rats exposed to local heating of the testes, IF levels of immunoactive inhibin remained unchanged from control values at 21-40 days after treatment, a finding confirmed by bioassay results. In rats made unilaterally cryptorchid for 10 months, levels of immunoactive inhibin in IF were reduced by 60% (P less than 0.01) in the abdominal compared with the contralateral scrotal testis.(ABSTRACT TRUNCATED AT 400 WORDS)