The process of testicular development that leads to initiation of spermatogenesis in bulls involves complex maturation mechanisms of the hypothalamus–pituitary–testes axis. Sexual development can be divided into three periods according to changes in gonadotropins and testosterone concentrations, namely the infantile, prepubertal, and pubertal periods. These changes are accompanied by changes in testicular cell proliferation and differentiation (Figure 4.1).

Infantile period

The infantile period is characterized by low gonadotropin and testosterone secretion and relatively few changes in testicular cellular composition. This period extends from birth until approximately 2 months of age in Bos taurus bulls.

Gonadotropin secretion during the infantile period is low due to reduced gonadotropin-releasing hormone (GnRH) 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 1 month of age.¹ 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 2 months of age.² 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.³ 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 1 and 4 months of age in bulls treated with naloxone, an opioid competitive receptor antagonist,⁴ 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.⁵

Direct evaluation of blood samples from the hypophyseal portal system has demonstrated that GnRH pulsatile secretion increases linearly from age 2 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 2 weeks, pulsatile LH secretion was not detected in jugular blood samples before 8 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 2 to 8 weeks of age without a concomitant increase in LH secretion may represent a reduced ability of the pituitary gland to respond to GnRH stimulus.³ 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.¹ 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,⁶ 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 2 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 1 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 2 months of age, but only Leydig cells formed postnatally are observed thereafter.^7,8 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 4 months of age. The number of undifferentiated Sertoli cells remains constant until 1 month of age, but cell multiplication is maximal between 1 and 2 months of age, decreasing thereafter until approximately 4 months of age. During the infantile period, the membranes of neighboring undifferentiated Sertoli cells contain few interdigitations and no special junctional complexes.^9,10 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 1 and 2 months of age.^9,11

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. It is not only associated with dramatic changes in testicular cellular composition, initial increase in testosterone secretion and timing of attainment of puberty, but also has long-lasting effects on testicular growth and sperm production. This period extends from approximately 2 to 6 months of age in Bos taurus bulls.

The early gonadotropin rise is driven by increased GnRH pulse secretion, as demonstrated by a dramatic increase in LH pulse frequency (Figure 4.2); 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 1 month to approximately 12–16 per day (one or more pulse every 2 hours) at approximately 4 months of age. Changes in pulse amplitude during this period are not consistent among reports; amplitude may be reduced, unchanged, or augmented.^1,12–16 LH-binding sites in testicular interstitial tissue have been demonstrated in bulls at birth and at 4 months of age and pulsatile LH secretion is an essential requirement for Leydig cell proliferation and differentiation and for maintenance of fully differentiated structure and function.¹⁷ Mesenchymal-like cells in the testes cease to proliferate around 4 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 4 to 7 months of age. Leydig cell numbers and mass per testis increase from 1 month (0.42 billion and 0.15 g/testis, respectively) to 7 months of age (6 billion and 5.8 g/testis, respectively), but mitosis after this age is rare.^7,8

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.¹⁸ 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 3–4 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 1–4 months of age to 7.5–9 pulses per 24 hours at 5 months of age.^19–22

The crucial role of the early gonadotropin rise (especially LH secretion pattern) 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 and FSH pulse frequency, pulse amplitude and mean concentrations at 3 months of age, delayed the peak mean LH concentration from 5 to 6 months of age, and reduced FSH and 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 2 hours to mimic pulsatile secretion from 1 to 1.5–2 months of age increased LH pulse frequency and mean concentration during the treatment period and resulted in greater scrotal circumference, testes weight, seminiferous tubules diameter, and number of germ and Sertoli cells in tubular cross-sections at 12 months of age.^23–25 The LH secretion pattern during the prepubertal period is also associated with age at puberty in bulls raised in contemporary groups, suggesting that this is the physiological mechanism by which genetics affect sexual development. Studies have shown that LH pulse frequency was greater around 2.5–5 months of age and that mean LH concentrations increased earlier and reached greater maximum levels in early- than in late-maturing Hereford bulls (age at puberty 9.5 and 11 months, respectively).^13,15

Additional support for the crucial role that the early gonadotropin rise plays in sexual development in bulls has been provided by studies demonstrating that nutrition during the prepubertal period affects LH secretion pattern, age at puberty, and testicular development. In one study in which bulls received different nutrition from 2 to 16 months of age, reduced LH pulse frequency during the prepubertal period resulted in delayed puberty in bulls receiving low nutrition, while a more sustained increase in LH pulse frequency in bulls receiving high nutrition was associated with hastened testosterone production and greater testes weight at 16 months of age when compared with bulls receiving low and medium (control) nutrition (Figure 4.3).¹⁶