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Regular Article |
-Subunit on Plasma Hormones and Testis Morphology in the Developing Cockerel1
a School of Animal and Microbial Sciences,
b The University of Reading, Whiteknights, Reading, Berkshire, United Kingdom RG6 6AJ
c School of Biological and Molecular Sciences, Oxford Brookes University, Oxford, United Kingdom OX3 0BP
ABSTRACT
Inhibins and activins are implicated as endocrine regulators of follicle-stimulating hormone production and of testicular steroidogenesis and spermatogenesis in mammals. The potential involvement of these proteins in cockerels was investigated by measurement of circulating inhibin A, inhibin B, total inhibin
-subunit immunoreactivity (ir-), activin A, LH, FSH, and testosterone from the juvenile state through to sexual maturity. Plasma inhibin A remained low between 6 to 12 wk of age and increased approximately threefold (P < 0.05) to a prepubertal peak between Weeks 14 to 18, followed by a gradual decline to the end of the study (Week 24). Although plasma FSH levels were not correlated to inhibin A before Week 16 (r = -0.17), they were negatively correlated from Week 18 (r = -0.49; P < 0.005). Inhibin B levels were below the assay detection limit until 16 wk of age but thereafter rose steadily in parallel with FSH (r = 0.27; P < 0.02) and testosterone (r = 0.35; P < 0.005). Thus, inhibins A and B showed divergent profiles during sexual maturation. Plasma ir- levels were much higher than dimeric inhibin levels throughout, although the relative difference varied with age. Plasma activin A levels were below the assay detection at all times. Juvenile cockerels were actively immunized against a synthetic chicken inhibin -subunit peptide conjugate to determine effects on plasma hormones and on testicular weight, morphology, and activin A content. Immunization generated circulating antibodies that bound 125I-bovine 32-kDa inhibin but did not affect plasma FSH or testosterone levels at any stage of development. However, immunization reduced postpubertal plasma LH levels (P < 0.05) and promoted increased testicular weight (24%; P < 0.01) and total testicular activin A content (42%; P < 0.001) at 24 wk. Testis weight of immunized birds was positively correlated with inhibin antibody titer (r = 0.61; P < 0.05). Live weight gain was not affected by immunization. Morphometric analysis of testis sections showed that inhibin immunization had no effect on the fractional volume of the seminiferous tubule wall, seminiferous tubule lumen, or interstitial tissue area. Likewise, seminiferous tubule surface area and surface area:volume ratios were not different from controls. These findings support differential roles for inhibins A and B in regulating the pituitary-testicular axis during sexual maturation in the cockerel but highlight the need for more detailed studies to distinguish between potential endocrine and local intragonadal roles of inhibin-related peptides and to elucidate the mechanism by which immunization against inhibin -subunit promotes testis enlargement without raising plasma FSH.
activin, FSH, inhibin, LH, puberty, testes, testosterone
INTRODUCTION
The relationship between the gonadally derived inhibins and pituitary FSH secretion has been well documented in several mammalian species but remains relatively obscure in birds, particularly in the male. Plasma immunoreactive inhibin -subunit (ir-) and FSH levels have been measured throughout cockerel development from the juvenile to adult stage and showed that ir- concentrations increased between 7 and 15 wk of age, whereas FSH increased from Week 11 to 29 [1]. However, there was no correlation between levels of the two hormones. Active immunization against inhibin -subunit has been used as an approach to investigate the physiology of inhibin in bulls and rams. Inhibin immunization increased plasma FSH but not LH or testosterone concentrations [2]. Testicular sperm density was also increased, but the weight of the testes and epididymis and total daily sperm production remained unchanged. Other studies also detected increased sperm production and plasma FSH but reduced plasma LH concentration in inhibin-immunized (IMM) bulls [3, 4]
In addition to having a likely endocrine role in the regulation of FSH, locally produced inhibins may also act as paracrine regulators of testicular function (review: [5]). Follistatin, inhibin/activin -, ßA- and ßB-subunit mRNA expression have been demonstrated in rat Sertoli cells, with expression varying at different stages of the seminiferous epithelial cycle [6, 7]. In prepubertal rat testis, the mRNA encoding the type IIB2 activin receptor was also localized in Sertoli cells around primary spermatocytes and meiotically dividing cells, suggesting that activin may have a function during meiotic division [8]. Intratesticular inhibin A treatment in rats also reduced the number of round spermatids without altering FSH levels [9].
To investigate the potential endocrine role of dimeric inhibins in the modulation of plasma FSH in the cockerel, the present study examined 1) the concentrations of inhibin A, inhibin B, and ir- in the circulation of normal cockerels and their interrelationship with plasma FSH, testosterone and LH from the juvenile state through to sexual maturity and 2) effects of active immunization of juvenile cockerels against inhibin -subunit on plasma FSH, LH, testosterone, and activin A levels and on testicular size, internal morphology, and activin A content. To assess the potential contribution of extragonadal tissues to circulating levels of inhibin-related proteins, various tissues were also collected from nonimmunized cockerels for measurement of their inhibin A, inhibin B, and activin A contents.
MATERIALS AND METHODS
Experimental Animals
Male chicks (ISA; n = 25) were raised in a floor pen until the onset of the experiment (41 days old), when chicks were caged individually. Birds were maintained on a standard short-day photoschedule of 8L:16D throughout the entire experimental period, with an ambient temperature of 21–23°C. Food and water were freely available at all times.
Experimental Design
At Day 41 (Week 5.9), the birds were weighed, and blood samples (2 ml) were collected by venipuncture of a brachial vein into heparinized tubes. Samples were centrifuged (3000 x g, 20 min), and the plasma was stored at -20°C until required. Live weights were recorded and blood samples were taken at ~2-wk intervals, between 1400 and 1600 h, until the end of the experiment (Week 24). At Day 65 (Week 9), birds were randomly allocated into one of three groups: nonimmunized controls (n = 6), carrier-immunized controls (n = 6), and IMM (n = 13). Inhibin-immunized cockerels were actively immunized against a synthetic peptide corresponding to the amino terminal region of the mature C subunit of chicken inhibin (amino acids 1–26, inclusive) conjugated (1:3 mass ratio) to tuberculin-purified protein derivative (PPD) using the glutaraldehyde method [10]. The peptide sequence was based on the deduced sequence from the cloned chicken inhibin -subunit cDNA [11] custom synthesized by Severn Biotech Ltd (Kidderminster, Worcs, UK). The primary injection consisted of 1 ml of emulsified immunogen conjugate (1 volume [100 µg] of immunogen to 2.2 volumes of Freund complete adjuvant) administered as 4 x 0.25 ml intramuscular injections at age 65 days (Week 9). Booster immunizations (B1-B4) consisted of 0.5 ml of emulsified immunogen conjugate (1 volume [50 µg] of immunogen to 2.2 volumes of Freund incomplete adjuvant), administered as 2 x 0.25 ml intramuscular injections at 78 days (Week 11), 93 days (Week 13), 119 days (Week 17), and 160 days (Week 23) of age.
At the same time, carrier-immunized cockerels (n = 6) were actively immunized against PPD. The primary injection consisted of 0.5 ml of emulsified PPD (1 volume [75 µg] of PPD to 2.2 volumes of Freund complete adjuvant). Booster immunizations (B1-B4) consisted of 0.5 ml of emulsified PPD (1 volume [50 µg] of PPD to 2.2 volumes of Freund incomplete adjuvant). Primary and booster PPD-control immunizations were administered as 2 x 0.25 ml intramuscular injections on the same immunization schedule as IMM birds. The nonimmunized controls (n = 6) received no injections or treatments. Comparisons between the untreated control group and the PPD-treated group were made as described in Statistical Analysis. In the absence of any significant differences, the results for the two groups were combined, and for the remaining part of this study, these birds are collectively termed control cockerels (CON; n = 12).
In Week 24, all cockerels were weighed, and terminal blood samples were taken before the birds were killed by intravenous injections of 200 mg pentobarbitone sodium. The testes were removed and weighed. A piece of testis (~1 cm3) was dissected and fixed in Bouin solution, embedded in paraffin wax, sectioned at 5 µm, and stained with hematoxylin and eosin. The remaining tissue was snap frozen on dry ice and stored at -70°C for further analysis. The brain, adrenal glands, and samples from liver, lung, heart ventricle muscle, and a piece of skeletal muscle were also removed, snap-frozen on dry ice, and stored at -70°C. A portion of each frozen tissue specimen was removed, weighed, and homogenized in a known volume of buffer A (PBS containing 1% [w/v] BSA and 0.1% [w/v] sodium azide), using an Ultra-Turrax T8 homogenizer (IKA, Staufen, Germany) to give the following volume/mass ratio homogenates: 1 x testis, 1.5 x brain and lung, 2 x liver and heart ventricle muscle, 3 x adrenal, and 3.5 x skeletal muscle. Homogenates were centrifuged at 3500 x g for 15 min, and the supernatants were stored at -20°C until further analysis. A 20-µl aliquot of homogenate was removed before centrifugation for DNA estimation by a fluorometric assay [12].
Assessment of Inhibin Antibody Titer
Plasma samples were tested for their ability to bind 125I-labeled bovine inhibin A (32 kDa) to provide a measure of antibody titer. Bovine inhibin A was isolated [13] and radiolabeled as described elsewhere [14]. Diluted (1:200; 400 µl) plasma samples were incubated with the tracer (approximately 10 000 cpm per tube; 50 µl) in a final volume of 450 µl for 24 h at room temperature. Separation of antibody-bound tracer was achieved using 200 µl of preprecipitated second-antibody reagent consisting of 50 mM PBS containing 50% (v/v) sheep anti-chicken IgG serum, 0.1% (v/v) normal chicken serum, and 3% (v/v) polyethylene glycol (Mr = 6000–7500). Tubes were mixed and incubated at room temperature for 45 min and centrifuged at 4000 x g for 30 min at 4°C. The supernatant was removed by aspiration, and the precipitated, antibody-bound 125I-labeled tracer was counted with a multigamma counter (LKB, Milton Keynes, Bucks, UK). After correcting for nonspecific binding (recorded in the absence of chicken serum/antiserum), results were expressed as percentage binding relative to the total counts per tube.
Immunoassays
Inhibin A, inhibin B, and activin A were determined using recently developed two-site ELISAs that employ monoclonal antibodies raised against synthetic peptide fragments of the human -, ßA and ßB subunits [15–17]. Recombinant human inhibin A, inhibin B, and activin A were used as assay standards, respectively. Assays were validated for use in the domestic fowl as described in the Results. The inhibin A and activin A two-site ELISAs have previously been validated for quantification of dimers in the hen ovary [18]. Total immunoreactive inhibin -subunit levels (ir-) were measured using a heterologous RIA employing a rabbit polyclonal antiserum against purified bovine inhibin [19] that has been validated previously for use in the domestic fowl [20]. This assay cross-reacts with dimeric inhibin forms and with monomeric inhibin -subunit forms including Pro-C. Bovine inhibin A (32 kDa) isolated in this laboratory [13] was used as an assay standard. Samples from IMM cockerels could not be assayed for inhibin A, inhibin B, and ir- because of interference from inhibin subunit-directed antibodies. Plasma samples were also assayed for testosterone [21], LH [22], and FSH [23] by RIAs, as described in the aforementioned publications.
Statistical Analysis
Confirmation of parallelism between assay standards and test sample dilution curves was made using linear regression analysis of transformed data. The log-log transformation was used to linearize the ELISA dose-response curves. Comparison of the slopes (95% confidence intervals) of the regression lines for standards and test samples indicated no significant departure from parallelism. This analysis was not possible when only the first two serial dilutions of a sample or extract gave a response above the detection limit.
One-way repeated measures ANOVA was used in conjunction with post hoc Fisher protected least significant difference (PLSD) test to determine whether hormone concentrations in carrier-immunized and control cockerels, CON and IMM cockerels, and inhibin antibody titers in the IMM cockerels varied at the different time points during development. Two-way repeated measures ANOVA was used to determine whether active immunization against inhibin -subunit significantly altered plasma concentrations of testosterone, LH, and FSH, with post hoc one-way ANOVA used to determine variation between IMM and CON plasma samples at individual time points. Post hoc tests were only performed when the repeated measures ANOVA yielded a significant F ratio. P < 0.05 was considered to be significant. Comparisons between carrier-immunized and control cockerels and between CON and IMM cockerels with respect to testicular wet weight and activin A content were made by one-way ANOVA. Unless otherwise stated, values are the means ± SEM.
Morphometric Image Analysis of Inhibin -Subunit Immunized and Control Cockerel Testis
The histology of testis tissue from IMM and CON cockerels was also investigated using a morphometric analysis method [24]. The test system used was made up of "probes" in the form of points and lines, and the test system was composed of a square test area (AT; 6z x 6.06z) that contained 21 lines of constant length, z, which were arranged in 7 equidistant and parallel rows, whereby the distance between the end-points of the lines was z in every direction. The total number of test points at the end of test line (P
) = 42, thus forming an equilateral triangular lattice. The test line z used in this study was equivalent to 137.7 µm. Sections of testes were analyzed and assessed for percentage fractional volume of seminiferous tubule wall, seminiferous tubule lumen, and interstitial area. Ten random fields of view per testis (5-µm sections stained with hematoxylin and eosin) were evaluated and used as criteria for the assessment of the effects of immunization. Tissue sections were encoded so that all analyses and statistical evaluations were done blind.
RESULTS
Validation of Inhibin A and Activin A Assays for Cockerel Plasma and Tissue Extracts
Serial dilutions of pooled plasma samples (juvenile and adult) and gonadal (testis) and extragonadal (lung, adrenal gland, liver, skeletal muscle, heart ventricle muscle, and brain) tissue extracts gave response curves in both inhibin A and activin A ELISAs that were parallel to the respective standard curve (see Figs. 1 and 2). Serial dilutions of IMM cockerel testes extract also gave a parallel response curve in the activin A ELISA (data not shown). The inhibin B content of extragonadal tissues was less than the minimum detection limit of the ELISA, whereas only the first two serial dilutions of pooled plasma samples gave a response above the detection limit. Serial dilutions of testis extract gave a dose-response curve parallel to the standard curve (see Fig. 1).
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Recovery of inhibin A, inhibin B, and activin A standard added into cockerel plasma and tissue samples was quantitative (means: inhibin A, 94%; inhibin B, 88% and activin A, 108%; n = 6 per sample). The detection limits of inhibin A, inhibin B and activin A ELISAs were 2, 15, and 50 pg/ml, respectively.
The cross-reactivities of a range of related substances in the inhibin A, inhibin B, and activin A ELISA have been reported previously [15–17] and were shown to be acceptably low. Within- and between-plate coefficients of variation were less than 10%.
Developmental Changes in Plasma LH, FSH, Testosterone, and Inhibin-Related Proteins in the Cockerel
The plasma profiles of inhibin A, FSH, LH, testosterone and ir- during sexual development of the cockerel are shown in Figure 3. There was no significant increase in plasma inhibin A from 6 to 12 wk of age. Levels rose over the following 2 wk (approximately threefold), with significantly higher plasma levels between 14 and 18 wk of age. Plasma inhibin A concentrations subsequently decreased from the peak at Week 18, with a significant fall by week 20 (58% decrease), followed by a further gradual decline over the next 4 wk. Ir- plasma levels were positively correlated with inhibin A, albeit weakly (r = 0.220; P < 0.05). A low concentration was present until 14 wk of age, then progressively rose 1.6-fold over the following 2-wk period. This level was sustained until Week 18, after which there was a significant transient decline at Week 20 (20% decrease) before concentrations rose again on Weeks 22 to 24. Plasma ir- concentrations were significantly higher than those of inhibin A and inhibin B at all times throughout development. The ir-: inhibin A ratio varied markedly during development, falling from ~200:1 in Week 6 to ~28:1 in Weeks 14–18. The ratio subsequently increased to ~200:1 on Week 24 as inhibin A levels declined. Plasma concentrations of inhibin B were less than the detection limit of the assay (12 pg/ml), until 16 wk of age, after which levels rose steadily (by approximately twofold) until the end of the experiment (Week 24).
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Plasma LH concentrations were relatively low between Weeks 6 to 12. Thereafter, levels rose to a peak on Week 16 (~40% increase; P < 0.05) before falling over the subsequent 2-wk period (23% decrease; P < 0.05) to a concentration that was maintained until Week 24. There was no correlation between plasma concentrations of LH and either inhibin A (r = -0.05; P > 0.05) or inhibin B (r = -0.22; P > 0.05). Plasma testosterone concentrations were very low until Week 12. A slight increase seen on Week 14 was followed by a progressive increase of approximately ninefold over the subsequent 6 wk, to Week 20, after which levels remained constant until Week 24. Plasma testosterone was positively correlated with levels of total ir- (r = 0.53; P < 0.001), inhibin B (r = 0.35; P < 0.005), and, to a lesser degree, inhibin A (r = 0.22; P < 0.05). Plasma FSH concentrations were very low until week 14, after which a progressive increase occurred until week 24 (5.5-fold increase). Although plasma FSH levels were not correlated with inhibin A before Week 16 (r = 0.17; P > 0.05), there was a strong negative correlation from Week 18 (r = -0.45; P < 0.005). In contrast, plasma FSH was positively correlated with inhibin B from Weeks 16 to 24 (r = 0.27; P < 0.02). Overall, plasma FSH was positively correlated with both ir- (r = 0.49; P < 0.001) and testosterone concentrations (r = 0.69; P < 0.001). Plasma activin A concentrations were below the detection limit of the assay (50 pg/ml) throughout the experiment.
Effects of Inhibin Immunization on Plasma LH, FSH, Testosterone, and Activin A During Cockerel Development
Figure 4 shows a comparison of plasma profiles of FSH, LH, testosterone, and inhibin antibody titer during sexual development of control cockerels and cockerels actively immunized against inhibin -subunit. All IMM cockerels responded to the immunization against inhibin -subunit with a significant increase in plasma 125I-labeled 32-kDa inhibin bovine inhibin-binding capacity (at 1:200 plasma dilution), which was maintained throughout the experiment. No binding of 125I-labeled 32-kDa inhibin was detected in plasma from CON cockerels. Analysis of the plasma testosterone and FSH profiles of CON and IMM cockerels showed no significant effect of treatment and no interaction between immunization and age (P > 0.05). Analysis of plasma LH concentrations over the entire experimental period (8–24 wk of age) with allowance for repeated measures indicated a significant reduction (P < 0.05) in IMM cockerels. Individual tests revealed significantly lower (P < 0.05) plasma LH concentrations in IMM cockerels at Weeks 16 (26% decrease), 18 (28% decrease), and 22 (28% decrease). Plasma activin A concentrations were below the detection limit of the assay at all times during development of both IMM and CON cockerels.
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Effects of Inhibin Immunization on Cockerel Growth Rates, Testis Weight, and Histology
Analysis of growth profiles showed no significant effect of immunization against -inhibin, with weights increasing ~4.5-fold from Weeks 6 to 24. The weight of the paired testes from immunized birds was significantly (P < 0.01) increased, to 44.08 ± 1.79 g (n = 13) from 35.47 ± 1.80 g (n = 12) in the controls at 24 wk of age, a 24% increase. Paired testes wet weight in IMM cockerels were positively correlated with mean (r = 0.580; P < 0.05) and with terminal inhibin antibody titers (r = 0.606; P < 0.05). Morphometric analysis of histological sections from CON and IMM testes showed that inhibin immunization had no effect on the fractional volume of the seminiferous tubule wall, seminiferous tubule lumen, or interstitial area. Likewise, seminiferous tubule surface area and surface area:volume ratios were not altered by immunization.
Effects of Inhibin Immunization on Testis Activin A Content
Active immunization against inhibin -subunit promoted a small but significant (14%; P < 0.05) increase in testicular activin A concentration (nanograms per milligram DNA) and total testicular activin A content (42%; P < 0.001). There was no significant correlation between mean or terminal inhibin antibody titers and testicular activin A content.
Gonadal and Extragonadal Production of Inhibin A, Inhibin B, and Activin A in the Cockerel
The distribution of inhibin A and activin A in gonadal and extragonadal (lung, adrenal gland, liver, skeletal muscle, heart ventricle muscle, and brain) tissues are shown in Figure 5. Inhibin A content in testis was significantly higher than in the other tissues studied, with concentrations decreasing, in rank order, from heart ventricle tissue to liver, brain, skeletal muscle, adrenal gland, and lung. Heart ventricle tissue contained markedly higher activin A concentrations than any other gonadal or extragonadal tissues examined. Activin A concentrations decreased, in rank order, from liver to skeletal muscle, adrenal gland, testis, brain, and lung. Inhibin B was not detectable in any tissue apart from testis, in which the level (4.9 ± 0.6 ng/mg DNA) was approximately 450-fold higher than that of inhibin A (11.2 ± 1.2 pg/mg DNA).
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DISCUSSION
This study reports the effects of active immunization against inhibin -subunit on reproductive hormone concentrations in the developing cockerel. A previous study has examined the effect of active immunization against the -inhibin subunit on egg production in quail [25], but we are not aware of any similar studies in male birds in the literature. The study also provides novel information on developmental changes in circulating inhibin A and inhibin B concentrations during sexual maturation and on the distribution of inhibin-related dimers in extragonadal tissues of the adult cockerel. Attempts to quantify activin A in peripheral plasma of cockerels were unsuccessful, concentrations being below the assay detection limit at all stages. Previous studies of plasma inhibin levels in the male chicken [1, 26] have utilized -subunit-directed RIAs, which lack the ability to discriminate between monomeric -subunit(s), inhibin A, and inhibin B. Because the chicken testis expresses , ßA, and ßB inhibin/activin subunits [27] it may be presumed that the tissue has the capacity to synthesize both inhibin A and inhibin B, in addition to the three forms of activin (A, AB, and B). Given the likelihood of differential testicular expression and assembly of the different subunits, it is evident that dimer-specific assays are required to establish the secretory profiles of the two inhibin forms during testis development.
The main findings with respect to circulating inhibin levels in normal control cockerels were as follows: 1) plasma inhibin A concentrations showed a marked though transient rise at 14–18 wk of age (onset of sexual maturity at ~18 wk), thereafter declining to low levels in the adult; 2) in contrast, plasma inhibin B concentrations were undetectable (<15 pg/ml) before 16 wk but then increased progressively after sexual maturation, in parallel with increasing FSH and testosterone; 3) from around the time of onset of sexual maturity, plasma inhibin A concentrations were negatively correlated with plasma FSH, whereas plasma inhibin B concentrations were positively correlated with FSH; 4) plasma concentrations of total ir- were relatively low from 6–14 wk, increased sharply between 14 and 16 wk, and, apart from a transient drop at 20 wk, remained high through to adulthood; and 5) total ir- concentrations were substantially higher than dimeric inhibin levels at all stages of development.
Plasma concentrations of ir- subunit are similar to those published previously by our laboratory [18] and by others [26] using the same assay. The use of different standards may explain differences in peptide levels in other studies [1]. Total ir--inhibin concentrations increased markedly between 14–16 wk, coincident with increased plasma concentrations of inhibin A and inhibin B. A similar increase in plasma ir--inhibin was reported in a White Leghorn-related strain maintained on long days (14L:10D) at 14–16 wk of age [26] but not in cockerels of the Babcock B300 strain maintained on short days (8L:16D) from 6–15 wk before transfer to long days (15L:9D) [1]. The reason for this difference is not clear but may reflect strain differences or the differing photoschedules used. In this study, the cockerels were raised throughout on short days (8L:16D), a photoschedule on which the birds reach puberty but are not photostimulated.
The transient increase in plasma inhibin A concentrations at 14–18 wk coincided with the prepubertal LH rise at 14–16 wk [1, 21, 28, 29] and with the first increase in plasma testosterone levels [21, 29]. This suggests that the role of inhibin A in regulation of the hypothalamic-pituitary-testis axis may be limited to a relatively short time period around the pubertal transition. What this role is remains to be investigated, but the evident lack of an inverse relationship with FSH, at least until after the onset of maturity, perhaps implies an intragonadal rather than systemic role. In this context, inhibin has been shown to enhance LH-induced androgen production from cultured rat interstitial/Leydig cells, whereas activin had the opposite effect [30]. Likewise, in chicken embryo testis culture, inhibin A enhanced, whereas activin A decreased, the conversion of pregnenolone to androgen [31].
Because the prepubertal peak in plasma inhibin A coincided with the rise (and subsequent fall) in LH but preceded the first increase in plasma FSH by two weeks, it is likely that LH is responsible for up-regulating testicular inhibin A production at this time. As yet, there is no information on the tissue distribution of inhibin/activin subunit mRNA and protein in the chicken testis, but responsiveness to LH implies that Leydig cells may synthesize inhibin A in the prepubertal cockerel, as suggested by several studies in mammals [32–34]. From the onset of sexual maturity at around 16 or 18 wk until the end of the study (24 wk), inhibin A and FSH were negatively correlated, whereas inhibin B and FSH were positively correlated; this suggests a changing contribution of inhibin A and inhibin B to the regulation of pituitary FSH secretion in the adult cockerel. Despite this, immunization against inhibin -subunit did not alter plasma FSH profiles at any stage during sexual maturation, which suggests either that inhibins A and B do not function as endocrine regulators of pituitary FSH secretion in the cockerel or that the titer and/or affinity of the antibodies produced was insufficient for effective immunoneutralization of circulating inhibins. The finding that inhibin immunization was associated with a significant reduction in plasma LH levels in the postpubertal period and a 25% increase in testis weight at 24 wk argues against the latter suggestion. Moreover, testis weight in immunized birds was positively correlated with plasma inhibin antibody titer, supporting the possibility of a causal relationship between inhibin antibodies and testis response. Our finding of increasing inhibin B levels after sexual maturation in the cockerel is consistent with recent reports that serum inhibin B increases after puberty in human males and is positively correlated with sperm production [35, 36].
Comparison of tissue contents of inhibin-related molecules in testis and extragonadal tissues from mature cockerels showed that inhibin A was most abundant in testis but was detectable in all other tissues examined, including adrenal, liver, heart, and brain. Inhibin B was only detected in testis, suggesting a specific gonadal source in the cockerel. Testicular activin A content was relatively low in comparison with other tissues. However, potential endocrine and/or intragonadal roles of activin A in the cockerel cannot be disregarded, although the functional significance of this remains to be established.
Active immunization of cockerels against inhibin -subunit did not alter plasma profiles of FSH or testosterone during sexual maturation, although plasma LH levels were significantly depressed from the time of onset of maturation. The effect of immunization on plasma LH cannot be explained by increased testosterone feedback, but it could possibly reflect a reduced action of inhibin on pituitary gonadotrophs. Inhibin has previously been shown to enhance GnRH-induced LH secretion from ovine pituitary cells in vitro [37] and to up-regulate expression of GnRH receptors [38]; a similar action of inhibin may pertain in birds. Plasma LH concentrations were also reduced in bulls immunized against inhibin -subunit; increased plasma FSH and testosterone levels were also recorded [4]. This suggests that in the bull, the increased plasma testosterone exerts a negative feedback effect on plasma LH. However, decreased plasma LH without a significant increase in plasma testosterone has also been reported in inhibin-immunized bulls [3]. These authors also found increased plasma FSH and oestradiol levels after immunization. It is possible that inhibin immunization increases testicular estradiol output in the cockerel. Estrogen treatment can reduce the responsiveness of chicken pituitary cells in vitro to GnRH [39], which reduces LH secretion. Thus, increased circulating estrogen concentration after inhibin immunization could explain the reduced LH secretion.
Immunization of cockerels against inhibin -subunit significantly increased the size and wet weight of the testes. However, morphometric analysis revealed that the increase in size was uniform throughout the testicular compartments. Although testicular function and morphology is critically dependent on pituitary gonadotrophins [40], the increased size of testes from immunized birds cannot be explained by changes in circulating FSH or increased LH in this study. Neither can the difference be explained simply by altered somatic growth rate because no differences in live weight were observed between immunized and control birds. Spermatozoa production is directly related to Sertoli cell number and testicular size in cockerels [41], and the increase in testicular wet weight shown here would probably also be reflected in an enhanced spermatozoa production rate, although measurement of sperm production rate was beyond the scope of the present study. Active immunization against inhibin -subunit in prepubertal male lambs caused a similar increase in testis diameter and increased daily sperm output in the adult [42] without significant changes in plasma FSH during the majority of the study. This contrasted with the observation that in IMM bulls, the enhanced sperm production, total sperm output, and sperm density was associated with increased plasma FSH [2–4]. The reason for this difference may be the state of sexual maturity at which the animals were immunized because compensatory growth of the remaining testis in hemicastrated cockerels only occurred if the surgery took place before mitotic division of the Sertoli cells was complete [41]. Therefore, it may be that the enhanced testicular growth demonstrated in the present IMM cockerels occurred before Sertoli cell mitosis was complete. Testicular activin A content was significantly raised in IMM cockerels, and activin stimulates Sertoli cell proliferation in rats [43]. A role of activin in testicular development was also suggested in activin receptor RII knockout mice that had a reduced seminiferous tubule volume [44]. Whether activin A has an analogous role in testicular development in the cockerel requires further investigation.
An alternative hypothesis to explain the increase in testicular size in inhibin -subunit-immunized cockerels is based on the demonstration that free inhibin -subunit precursors can compete with FSH for binding its receptor [45]. It is evident from the present data that cockerel testes produce large amounts of free inhibin -subunit; this may act locally to reduce Sertoli cell responsiveness to FSH. Immunization against inhibin -subunit may thus enhance the sensitivity to FSH, which in turn could promote cell proliferation leading to increased testis size. However, further work is required to identify and characterize inhibin subunit precursor forms secreted by cockerel testis and to demonstrate any putative modulatory actions on FSH-receptor interaction.
In conclusion, active immunization of juvenile cockerels against inhibin -subunit promotes an increase in testis size through a mechanism that does not depend on raised FSH secretion. Immunization may perturb the regulatory mechanisms that fix the number of Sertoli cells at a particular stage of development. Possible mechanisms to enhance testicular size may involve an increased responsiveness of the testes to circulating FSH and/or an increased proliferation of Sertoli cells in response to enhanced testicular activin A concentration.
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ACKNOWLEDGMENTS
We thank Dr. J. Proudman (United States Department of Agriculture, Beltsville, MD) for providing chicken FSH assay reagents, Dr. A.F. Parlow for providing recombinant human inhibin A and activin A, and Mr. S.A. Feist for technical assistance.
FOOTNOTES
First decision: 29 October 2000.
1 This work was supported by the Biotechnology and Biological Sciences Research Council. 
2 Correspondence. FAX: 118 931 0180; r.t.gladwell{at}reading.ac.uk
Accepted: February 29, 2000.
Received: October 4, 1999.
REFERENCES
-subunit messenger RNA expression in the rat seminiferous epithelium. Mol Cell Endocrinol 1991; 82:165–173.[CrossRef][Medline]-subunit: effects on gonadotropin concentrations and ovulation rate in sheep. J Endocrinol 1990; 124:167–176.-subunit of inhibin from chicken ovarian granulosa cells. Biol Reprod 1993; 49:453–458.[Abstract] subunit and its isolation from bovine follicular fluid. J Mol Endocrinol 1989; 2:189–200.-subunit levels in granulosa and thecal layers of developing preovulatory follicles in the chicken. Endocrinology 1998; 139:1164–1171.-subunit expression in rat leydig cell cultures. Mol Cell Endocrinol 1989; 66:119–122.[CrossRef][Medline]-inhibin modulate follicle-stimulating hormone receptor binding and biological activity. Endocrinology 1991; 129:1987–1999.This article has been cited by other articles:
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  T. M. Lovell, P. G. Knight, N. P. Groome, and R. T. Gladwell Changes in Plasma Inhibin A Levels During Sexual Maturation in the Female Chicken and the Effects of Active Immunization Against Inhibin {{alpha}}-Subunit on Reproductive Hormone Profiles and Ovarian Function Biol Reprod, January 1, 2001; 64(1): 188 - 196. [Abstract] [Full Text]
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), inhibin B (
), FSH (
), LH (
), and testosterone (
) during sexual development of the cockerel. Values are means + SEM (n = 12). dl corresponds to minimum assay detection limit. Significant differences (P < 0.05) between hormone concentrations at different time points are indicated by different letters.
) measured in immunized birds are also shown. Values are means ± SEM (n = 12, control; n = 13, immunized). Significant differences (P < 0.05) between antibody titers at different time points are indicated by different lower case letters and between-groups differences in hormone concentration by different capital letters. The ages at which primary (P) and booster (B1–B4) immunizations were given are indicated.
