Inhibin-A and Inhibin-B in stallions: Seasonal changes and changes after down-regulation of the hypothalamic-pituitary-gonadal axis

Highlights

• Inhibin-B is the major inhibin heterodimer present in stallion blood.

• Both inhibin-A and inhibin-B are correlated with photoperiod with seasonal peaks corresponding to long day lengths.

• Down-regulation of the pituitary axis with a GnRH antagonist decreased concentrations of inhibin-B but not inhibin-A.

• Testis volume was strongly correlated with inhibin-B but not inhibin-A concentrations.

Abstract

The biological function of inhibin is mediated by two heterodimers, inhibin-A and inhibin-B. The relative importance of inhibin-A and –B in male reproductive function varies considerably across species with inhibin-B predominating in many species, whereas inhibin-A appears relatively more important in rams. Research reported to date in stallions has examined total or immunoreactive (ir) inhibin which does not distinguish the two heterodimers. Therefore, the objective of this study was to characterize changes in inhibin-A and inhibin-B concentrations in stallions: 1) across season for a period of one year, and 2) after downregulation of the hypothalamic-pituitary-gonadal (HPG) axis. In Study one, serum samples were obtained monthly from five stallions for a period of one year. Serum concentrations of inhibin-A, inhibin-B, testosterone and estrone sulfate were determined by ELISA. In Study two, stallions were treated with the GnRH antagonist, acyline (n = 4; 330 mg/kg acyline IM) or vehicle control (n = 4; vehicle alone) every five days for 50 days. Plasma concentrations of inhibin-A and –B were determined by ELISA at Days 0, 6, 12, 22, 37, 59, 80, 87 and 104 after initiation of acyline treatment. Testis volume was determined by ultrasonography at weekly intervals. In Study 1, both inhibin-A and inhibin-B showed seasonal changes in concentration with highest concentrations in increasing day length and lowest concentrations in short day lengths. Inhibin-B (overall mean 107.8 ± 4.1 pg/mL) was present at 4.7-fold higher concentrations in serum than inhibin-A (overall mean 23.0 ± 0.7 pg/mL). In Study 2, plasma concentrations of inhibin-B but not inhibin-A were significantly downregulated by administration of the GnRH antagonist, acyline. When the HPG axis was downregulated by acyline, testis volume was strongly correlated with inhibin-B (r = 0.73; P < 0.05) but not inhibin-A (r = 0.22; P = 0.20). In summary, inhibin-B appears to be the predominant form of inhibin in the stallion which undergoes seasonal regulation along with other reproductive parameters and is co-regulated with other endocrine parameters of the HPG axis.

Introduction

Inhibins are members of the TGFβ superfamily and are heterodimers that are composed of a common α subunit linked with either a βA subunit (inhibin-A) or a βB (inhibin-B) subunit [1]. A variety of molecular forms of inhibin are present in circulation, but bioactivity appears limited to inhibin-A and –B [1,2]. Gonadal production of these glycoprotein hormones acts in a negative feedback loop to reduce the secretion of follicle stimulating hormone (FSH) by the pituitary [3,4]. In turn, FSH appears to stimulate inhibin secretion by the testis, completing the feedback loop. Therefore, in normal adult males, circulating inhibin concentrations are negatively correlated with FSH concentrations [5].

Although inhibin is thought to be principally a product of the Sertoli cell in the testis, immunolocalization of inhibin α and βB subunits in the testis suggests that Sertoli cells, Leydig cells and possibly germ cells may be involved in inhibin formation [1,5,6]. Studies to date have not characterized specific bioactive forms of peripheral inhibin in stallions; however, it has been reported that inhibin α, βA and βB subunits were expressed in Sertoli and Leydig cells of the mature stallion and that inhibin-B was likely the major form present in stallions based upon relative tissue concentrations of inhibin-A and −B in stallion testis [4,6].

Generally, inhibin-B appears to be the major inhibin isoform in males [7]. In human and nonhuman primates, Sertoli-cell production appears to be limited to inhibin-B, and inhibin-B represents the major circulating form [8,9]. Correspondingly, inhibin-A is undetectable in the serum of adult men [1]. Inhibin-B is also the major circulating isoform in rats [10], hamsters [11], and miniature pigs [12]. In contrast, only inhibin-A is present in circulation of rams [13], and inhibin-A and –B are present in circulation in bulls [14]. Specific inhibin isoforms have not been characterized in circulation in stallions; however, a recent report in mares identified inhibin-A as the physiologically relevant isoform [15].

Early immunoassays for inhibin were based upon a 31kD bovine inhibin isolated from follicular fluid, and these assays provided a critical resource for description of the biology of inhibin [16]. Unfortunately, these immunoassays were unable to discern the bioactive heterodimers present in serum because the assay recognized only the inhibin α subunit [1,3]. It was the development of monoclonal antibodies to inhibin α and β subunits which allowed the development of sandwich ELISAs which distinguished inhibin-A and -B [1,8,17,18]. These immunoassays have recently been validated for detection of peripheral inhibin-A and inhibin-B in horses based upon specific monoclonal antibodies to either inhibin βA or βB as capture antibodies and a specific monoclonal antibody for detection of inhibin α in a sandwich ELISA [15]. To date, existing publications which have examined changes in circulating inhibin in stallions have relied upon total inhibin immunoassay, and therefore have been unable to distinguish active heterodimers in circulation [4,6,19,20]. The lack of specific immunoassays for inhibin-A and –B has precluded investigation of the relative physiologic or clinical relevance of these two molecular forms in the stallion.

Serum inhibin concentrations have been examined as a marker of male fertility in several animal species and in humans [1,3,21]. Serum inhibin-B concentrations are associated with active spermatogenesis and may reflect Sertoli-cell function in men [1,22]. Arrest of spermatogenesis is associated with decreased concentrations of inhibin-B, and serum inhibin-B concentrations are correlated with testis volume and sperm number [[23], [24], [25]]. Concentrations of serum inhibin-B were strongly correlated with total sperm number [26], and inhibin-B appeared to be a reliable marker for human male fecundity [27]. In stallions, immunoreactive (ir-)inhibin concentrations were reduced in infertile males although the assay used for this study was unable to distinguish the heterodimers [28].

The stallion reproductive axis is influence by photoperiod, and seasonal changes in circulating ir-inhibin have been characterized in the stallion [6,28,29]. Both Taya et al. [6] and Nagata et al. [4,29] demonstrated increased ir-inhibin in stallions during the breeding season associated with increases in testosterone, estradiol, and gonadotropins [29]. Again these studies utilized immunoassays which did not distinguish between the two heterodimers, and the relative importance of circulating concentrations of inhibin-A and –B and seasonal influences on these heterodimers in the stallion remains unreported.

Regulation of inhibin secretion in the stallion is poorly understood. In humans, FSH stimulates the physiologically important inhibin-B secretion by the testis via stimulation of the inhibin α precursor, pro-α-C [8]. Both ir-inhibin and inhibin pro-αC, a precursor of inhibin α, were rapidly downregulated in stallions treated with anabolic steroids [19,30] reflecting a downregulation of the hypothalamic-pituitary-gonadal (HPG) axis. Recently, we reported on the effects of downregulation of the HPG axis in stallions using a GnRH antagonist, acyline [31] with corresponding decreases in gonadotropins, testosterone and estrone sulfate in treated stallions. Here, we further characterize associated changes in inhibin heterodimers subsequent to downregulation of the HPG axis of stallions. Therefore, the objectives of the current study were to define inhibin isoform secretion in stallions, examine seasonal changes in serum inhibin-A and –B concentrations and examine changes in inhibin-A and –B concentrations after downregulation of the HPG axis with a GnRH antagonist (acyline).