secretion; maturation changes within the hypothalamus result in increased GnRH pulse secretion and drive the transition from the infantile period. Increased GnRH secretion is dependent on either the development of central stimulatory inputs or removal of inhibitory inputs. Hypothalamus weight and GnRH content do not increase during the infantile period, but hypothalamic concentrations of estradiol receptors decrease after one month of age [7]. However, the hypothesis that GnRH secretion is low during infancy due to elevated sensitivity of the hypothalamus to the negative feedback of sex steroids (gonadostat hypothesis) has been questioned in bulls, since castration does not alter luteinizing hormone (LH) pulse frequency or mean concentrations before two months of age [8]. Nonetheless, since GnRH secretion into hypophyseal portal blood is not necessarily accompanied by LH secretion during the infantile period, experiments that use LH concentrations to infer GnRH secretion patterns during this period need to be interpreted with caution [9]. Another possibility is that removal of opioidergic inhibition and/or increased dopaminergic activity may be involved in triggering the increase in GnRH secretion during the infantile period. Opioidergic inhibition of LH pulse frequency during the infantile period has been demonstrated by increased LH secretion between one and four months of age in bulls treated with naloxone, an opioid competitive receptor antagonist [10], whereas concentrations of norepinephrine, dopamine, and dopamine metabolites increased twofold to threefold in the anterior hypothalamic–preoptic area in bulls aged 0.5–2.5 months [11].
Direct evaluation of blood samples from the hypophyseal portal system has demonstrated that GnRH pulsatile secretion increases linearly from age two weeks (3.5 pulses per 10 hours) to 12 weeks (8.9 pulses per 10 hours) in bulls. Although GnRH secretion into hypophyseal portal blood was detected at two weeks, pulsatile LH secretion was not detected in jugular blood samples before eight weeks of age. In addition, GnRH pulses are not necessarily accompanied by LH secretion until 8–12 weeks of age, when all GnRH pulses result in LH pulses. The increase in pulsatile GnRH release from two to eight weeks of age without a concomitant increase in LH secretion may represent a reduced ability of the pituitary gland to respond to GnRH stimulus [9]. The period in which GnRH pulses do not stimulate LH secretion correspond to a period during which there is an increase in pituitary weight, GnRH receptor concentration, and LH content [7]. Moreover, frequent GnRH treatments during the infantile period in calves increases pituitary LH‐β mRNA, LH content, and GnRH receptors, with resulting increases in LH pulse frequency and mean concentrations [12], indicating that increased GnRH pulse frequency results in increased pituitary sensitivity to GnRH. With time, the increased GnRH secretion results in the increased LH pulse frequency observed during the prepubertal period.
From birth until approximately two months of age, mesenchymal‐like cells comprise the majority of the cells in the testicular interstitial tissue. Typical Leydig cells constitute about 6% of all intertubular cells at one month of age and a number of these cells are found in an advanced degenerative state, probably as remnants of the fetal Leydig cell population. Degenerating fetal and newly formed Leydig cells coexist until two months of age, but only Leydig cells formed postnatally are observed thereafter [13, 14]. The diameter of the seminiferous tubules is approximately 50 μm during the infantile period; tubule is actually a misnomer, since these are in fact solid cords with no lumen at this stage of development. Undifferentiated Sertoli cells (or undifferentiated supporting cells) are the predominant intratubular cells from birth until approximately four months of age. The number of undifferentiated Sertoli cells remains constant until one month of age, but cell multiplication is maximal between one and two months of age, decreasing thereafter until approximately four months of age. During the infantile period, the membranes of neighboring undifferentiated Sertoli cells contain few interdigitations and no special junctional complexes [15, 16]. These immature Sertoli cells produce and secrete anti‐Müllerian hormone (AMH) and circulating concentrations increase until two months of age [1]. The germ cell population is composed solely of gonocytes (or prespermatogonia) at birth. Gonocytes are usually centrally located and have a large nucleus (~12 μm in diameter) with a well‐developed nucleolus. Gonocyte proliferation slowly resumes between one and two months of age [15, 17].
Prepubertal Period
The prepubertal period is characterized by a temporary increase in gonadotropin secretion, the so‐called early gonadotropin rise. The early gonadotropin rise is a critical event in the sexual development of bulls. Not only is it associated with dramatic changes in testicular cellular composition, initial increase in testosterone secretion, and timing of attainment of puberty, but also it has long‐lasting effects on testicular growth and sperm production. This period extends from approximately two to six months of age in B. taurus bulls.
The early gonadotropin rise is driven by increased GnRH pulse secretion, as demonstrated by a dramatic increase in LH pulse frequency (Figure 5.2); pulsatile discharges of follicle‐stimulating hormone (FSH) have been observed in bulls but are much less evident than those of LH. The number of LH pulses increases from less than one per day at one month to approximately 12–16 per day (one or more pulses every two hours) at approximately four months of age. Changes in pulse amplitude during this period are not consistent among reports; amplitude may be reduced, unchanged, or augmented [2, 7,18–21]. LH‐binding sites are present in testicular interstitial tissue at birth and at four months of age and pulsatile LH secretion is an essential requirement for Leydig cell proliferation, differentiation, and maintenance of fully cell differentiated structure and function [22]. Mesenchymal‐like cells in the testes cease to proliferate around four months of age and start to differentiate into contractile myofibroblasts and Leydig cells. Differentiating, mitotic, and degenerating Leydig cells are observed in close proximity from four to seven months of age. Leydig cell numbers and mass per testis increase from one month (0.42 billion and 0.15 g/testis, respectively) to seven months of age (6 billion and 5.8 g/testis, respectively), but mitosis after this age is rare [13, 14]. Circulating insulin‐like peptide 3 (INSL3), a major product and biomarker of Leydig cell functional capacity, increases linearly between two and six months in bulls [6, 23].
Figure 5.2 Serum LH concentrations between 10 and 30 weeks of age in Angus and Angus × Charolais bulls. Graphs closely exemplify the mean pulse frequency and pulse amplitude observed in bulls receiving adequate nutrition.
Sources: [2, 4, 6].
The characteristic pulsatile nature of LH secretion is important for testosterone production, since continuous exposure of Leydig cells to LH results in reduced steroidogenic responsiveness due to downregulation of LH receptors [24]. Initiation of Leydig cell steroidogenesis is characterized by increased androstenedione secretion, which decreases as the cells complete maturation and begin secreting testosterone. During the first three to four months of age, testosterone concentrations are low and secretion does not necessarily accompany LH pulses. After this age, LH pulses are followed by testosterone pulses and mean testosterone concentrations begin to increase. The number of testosterone pulses increases from 0.3–2.3 pulses per 24 hours at one to four months of age to 7.5–9 pulses per 24 hours at five months of age [25–28].
The crucial role of the LH secretion pattern during the early gonadotropin rise in regulating sexual development in bulls has been demonstrated in several studies using a variety of approaches. Prolonged treatment with a GnRH agonist in calves aged 1.5–3.5 months decreased LH pulse frequency, pulse amplitude, and mean concentrations at three months of age, delayed the peak mean LH concentration from five to six months of age, and reduced testosterone concentrations between 3.5 and 4.5 months of age. These hormonal alterations were associated with delayed puberty and reduced testes weight and number of germ cells in tubular cross‐sections at 11.5 months of age. On the other hand, treatment with GnRH every two hours to mimic pulsatile secretion from 1 to 1.5–2 months of age increased LH pulse frequency