The scrotum and spermatic cords

The scrotum is composed of an outer layer of thick skin and three underlying layers, the tunica dartos, the scrotal fascia, and the parietal vaginal tunica. The scrotal skin is extensively populated with numerous large adrenergic sweat and sebaceous glands that are highly endowed with thermal receptors and nerve fibers. Neural stimulation from the thermal receptors enables the tunica dartos, which consists of smooth muscle fibers and lies just beneath the scrotal skin, to contract and relax in response to changes in temperature gradients and facilitates the cooling of the scrotal surface via scrotal glandular sweating.¹⁹ Thus the scrotum plays an important role not only in housing and protecting the testes but also has a role in thermoregulation of the testes. The spermatic cord connects the testes to the body and provides access to and from the body cavity for vascular, neural, and lymphatic systems that support the testes. In addition, the spermatic cord accommodates the cremaster muscle, the primary muscle supporting the testes, and the pampiniform plexus, a complex and specialized venous network that wraps around the convoluted testicular artery.²¹ This vascular arrangement is very important in temperature regulation of the testicular environment. The plexus consists of a coil of testicular veins that provide a counter-current temperature exchange system: this is an effective mechanism whereby warm arterial blood entering the testes from the abdomen is cooled by the venous blood leaving the testes. Testicular arteries originate from the abdominal aorta and elongate as the testis migrates into the scrotum.¹⁹ In cattle and other large domestic ruminants these arteries are highly coiled, reducing several meters of vessel into as little as 10 cm of spermatic cord.¹⁹ The arterial coils and venous plexus are complex structures that form during fetal life in cattle.^19,22 Because of the pendulous nature of the bovine scrotum, testicular cooling is facilitated by the contraction and relaxation of the cremaster muscle, which draws the testes closer to the abdominal wall during cooler ambient temperatures and vice versa during warmer temperatures. Figure 2.1 shows bright-field and thermal images of the bovine testes that demonstrate the change in temperature from the neck to the tip of the scrotum as the testes thermoregulate during elevated environmental temperatures. Scrotal and testicular thermoregulation is a complex process involving a number of local mechanisms that strive to maintain the testes at environmental and physiological conditions conducive for normal spermatogenesis. For additional reading on testicular thermoregulation in the bull the reader is referred to the review by Kastelic et al.²³ and Chapter 3 of this book.

Interstitial tissue (Leydig cells)

Franz Leydig, a German zoologist, first described the interstitial cells of the testes in 1850 and these cells have since been known as Leydig cells. The Leydig cells reside in the interstitial tissue of the testis, a meshwork of loose connective tissue filling the spaces between the seminiferous tubules and blood vessels. In mammalian testes, the Leydig cells occur mainly as clusters in the angular interstices between the seminiferous tubules and are closely associated with the walls of small arterioles.^8,24 The Leydig cell content of testes varies from species to species. The Leydig cells are thought to be the principal source of androgens in the testis. The development of the Leydig cells, via metamorphosis of mesenchymal precursor cells, has been observed to be continuous throughout life after the time of puberty in the bull.²⁵ Christensen²⁶ provides a very detailed and interesting review of the history of Leydig cell research dating from Leydig’s description of the cells in the 1850s to the confirmation provided in the mid-1960s that these cells were indeed primarily responsible for testicular androgen synthesis and secretion. There is extensive evidence to suggest that early fetal Leydig cells are steroidogenically active in some mammalian species including the pig²⁷ and sheep.²⁸

The Leydig cells of most mammalian species studied are basically similar, with some minor variations in appearance, size, and the relation of Leydig cell clusters to the lymph or blood vessels of the interstitial tissue. Some variation in the extent of cytoplasmic structures exists, but ultrastructurally Leydig cells show considerable overall similarity.⁸ Fawcett et al.²⁹ described in detail the morphology of interstitial tissue of several mammalian species, and categorized three groups based on the abundance of Leydig cells and the relationship between volume of intertubular lymph structures and connective tissue. In the first group are the guinea-pig and rodents (rat, mouse). In these species only 1–5% of the testicular volume is occupied by Leydig cells, for example 2.8% in the rat.³⁰ The bull, monkey, elephant, and human fall into the second group. In these species, the connective tissue of the interstitium is very loose and the Leydig cells are scattered throughout the interstitium and are closely associated with a well-developed lymph system. The Leydig cells comprise only a small portion of the testicular volume (~15%).⁸ In the third group are the domestic boar and horse. In these animals there is abundant interstitial tissue packed with Leydig cells (20–60% of testicular volume).³¹ The reason for the high density of Leydig cells in these species is not known, but Parkes¹⁰ and Fawcett et al.²⁹ have attributed this phenomenon to the vast amounts of estrogens produced by the boar and stallion and the large quantities of musk-smelling 16-androstenes secreted by the boar testes.³² For more detailed discussions on the cytology of Leydig cells the reader is referred to an excellent chapter by de Kretser and Kerr.³³

Hypothalamic–pituitary hormone regulation of the testis

Hormone action depends on the release of hormones from the appropriate endocrine gland and transportation via the vascular circulatory system to the target tissue where the hormone binds to cellular receptors, thus inducing a physiological response. In some cases these receptors are very hormone-specific. The response at the target tissue may depend on the level of receptor expression and concentration of hormone. Some hormones regulate their own receptors, others may require synergism between two hormones, and others still may have their receptors regulated by other hormones.³⁴ The general characteristics of neuroendocrine regulation of the mammalian testis by the hypothalamic–pituitary axis are well established,³⁵ but in some species this may be seasonally regulated. Domestic cattle are considered to be continuous or nonseasonal breeding species.³⁶ However, there is information in the literature to indicate that domestic beef and dairy bulls have a functional hypothalamic–pituitary–gonadal axis that is seasonally regulated and thus may influence levels of gonadal steroidal and germ cell production. In a study using composite breeds of mature bulls, Stumpf et al.³⁷ observed that season of the year influenced the profile of gonadotropins in the circulation of both gonadectomized and intact males, and that the greatest secretion of gonadotropins occurred at the spring equinox. In addition, these authors observed that season of the year also influenced secretion of testosterone in intact males, but that more testosterone was released in response to LH during the time of the summer solstice. Others have reported similar effects, where location affected reproductive traits of bulls including semen quality and blood concentrations of LH and testosterone.^38,39 In addition, sensitivity in responsiveness to exogenous gonadotropin administration and testosterone secretion in bulls was observed to be seasonally influenced.⁴⁰

The regulation of testicular function by hormonal mechanisms depends on the integrated actions of gonadotropins, such as LH, follicle-stimulating hormone (FSH) and prolactin, and steroids (androgens and estrogens) on the Leydig cell.⁴¹ Gonadotropin-releasing hormone is the primary hypothalamic hormone governing the regulation of the synthesis and release of the gonadotropins LH and FSH by the anterior portion of the pituitary gland. LH is primarily responsible for testosterone production by the Leydig cells while FSH facilitates Sertoli cell proliferation and support of the germinal cells. Although it has been well established that gonadotropins stimulate testicular function, it was during the 1970s that the basic mechanism and site of action in the testis were identified. LH has been shown to be the gonadotropin essential for the maintenance of testicular testosterone production.^41,42 Catt and Dufau⁴³ have reviewed the mechanisms of action of the gonadotropins and concluded that they all elicit target cell response by similar mechanisms. In fact, LH provides the most important physiological regulation of the production of androgens by the Leydig cells of the testis.^44,45

Leydig cells contain plasma membrane-bound receptors that specifically bind LH. The binding of LH stimulates adenyl cyclase to produce cyclic adenosine monophosphate (cyclic AMP), a second messenger in the cell cytoplasm, which in turn activates cyclic AMP-dependent protein kinase thereby increasing the conversion of cholesterol to pregnenolone. The action of LH can be readily demonstrated in hypophysectomized and intact males. Removal of the pituitary gland is followed by rapid cessation of testosterone production, loss of enzymes involved in steroidogenesis, and testicular atrophy.⁴⁶ There is little direct evidence to support a role of FSH in Leydig cell steroidogenesis, but there is some evidence indicating that this gonadotropin may play a function in the conversion of androgens to estrogens in the Sertoli cells.⁴⁷ Bartke et al.⁴² have suggested that FSH may act on the aromatizing enzyme system of testosterone biosynthesis, but little evidence exists for action on other steps of the steroidogenesis pathway in the testis. In bulls, testosterone secretion is not tonic, but is characterized by episodic pulses dictated by the release of luteinizing hormone releasing hormone (LHRH) from the hypothalamus and LH from the anterior pituitary gland.⁴⁸ Schanbacher⁴⁸ reports that a temporal relationship exists between concentrations of LH and testosterone in the blood, and that there is evidence that episodic secretion depends on discrete episodes of LHRH discharge from the hypothalamus.

Prolactin

Prolactin (PRL) is known to enhance the effect of LH on spermatogenesis. The physiological importance of PRL in the regulation of Leydig cell function was first determined using three animal models:^41,42 the golden hamster, where testicular regression can be induced by short photoperiod; hypophysectomized mice; and the hereditary dwarf mouse with congenital PRL deficiency and infertility. In all three models there is a deficiency in plasma concentrations of PRL and testosterone and testicular atrophy is evident. PRL therapy, on the other hand, stimulates spermatogenesis in the dwarf mouse and restores testicular function in the golden hamster; in hypophysectomized animals, PRL, in the presence of LH, induces spermatogenesis and restores plasma testosterone. In men, PRL has an important role in the control of testosterone and reproductive function.⁴⁹ At very high concentrations (hyperprolactinemia), PRL has an antigonadal effect in men, inhibiting testicular function, and is associated with hypogonadism.⁵⁰

Other regulatory factors

The effects of other regulatory factors, such as growth hormone (GH), on testicular steroidogenesis have not been fully clarified. However, in view of the structural similarities between PRL and GH, it is thought that both hormones may have comparable effects on Leydig cell function.⁴² The effect of GH may be more significant during puberty (see Chapter 4). However, administration of gonadal steroids to intact animals is known to cause reduced androgen production, an effect that has been attributed to inhibition of gonadotropin secretion with secondary effects on testicular endocrine function. Estrogen receptors have also been detected in interstitial tissue of the testis and since decreased testosterone levels are not correlated with a corresponding change in plasma LH, some authors suggest that estrogen may exert a direct inhibitory action on Leydig cell function.^{51, 52} The effects of corticosteroids on Leydig cell function have been noted. Boars treated with adrenocorticotropin (ACTH) experienced increased testicular testosterone simultaneously with increased secretion of adrenal corticosteroids.⁵³ A transient increase in peripheral circulation of both corticosteroids and testosterone was first observed in boars following acute treatment with ACTH while a decrease in testosterone occurred following chronic treatment.⁵⁴ These authors concluded that prolonged stressful conditions may lead to chronic elevation in ACTH levels, thus suppressing testosterone production and inducing poor breeding activity.

In reviewing the literature, Tilbrook et al.⁵⁵ have determined that stress-related influences on reproduction is complicated and not fully understood, but the process may involve a number of endocrine, paracrine, and neural systems. The significance of stress-induced secretion of cortisol varies with species. In some instances, there appears to be little impact of short-term increases in cortisol concentrations and protracted increases in plasma concentration seem to be required before any deleterious effect on reproduction is apparent. In a comprehensive review, Moberg⁵⁶ describes the influence of the adrenal axis on gonadal function in mammals. Subsequently, the same author published a detailed review⁵⁷ of the impact of behavior on domestic animal reproductive performance and placed particular emphasis on how intensive livestock management practices can contribute to stress-induced disruption of normal reproductive function. However, the emphasis has primarily been on studies investigating the effects of stress-induced ACTH release in females.

Elevated plasma ACTH results in increased adrenal corticosteroid synthesis and under chronic conditions may inhibit gonadotropin-releasing hormone and gonadotropin hormone production, thereby reducing gonadotropin (LH) release from the pituitary gland and thus impacting normal ovarian function in females. Nevertheless, studies have shown that stress-induced elevation in ACTH affects males in a physiologically similar manner by impacting gonadotropin regulation of testicular function. Liptrap and Raeside⁵⁸ observed the effects of cortisol on gonadotropin-releasing hormone in boars, while Matteri et al.⁵⁹ observed that stress or acute ACTH treatment suppresses gonadotropin-releasing hormone-induced LH release in the ram. In Holstein bulls treated with a bolus of ACTH (0.45 IU/kg), plasma LH or FSH concentrations did not appear to be negatively affected, but it did appear to reduce plasma testosterone concentrations.⁶⁰ Pharmacological concentrations of ACTH (200 IU every 8 hours) over a 6-day period resulted in reduced plasma testosterone in yearling and mature bulls within 8 hours and 4 days of initial treatment, respectively, and this decrease persisted in both age groups for an additional 24 hours after the last ACTH injection.⁶¹ While semen viability, concentration, and sperm output were unaffected by the prolonged ACTH treatment concomitant with a subsequent marked increase in glucocorticoids and decrease in testosterone, a small increase in semen content of immature sperm or sperm with abnormal heads was observed.⁶¹ Thus, it appears that under prolonged stressful conditions, gonadotropin secretion is more likely to be suppressed thus inhibiting reproductive performance. In addition, studies have demonstrated that glucocorticoids can inhibit gonadotropin secretion in some circumstances. Tilbrook et al.⁵⁵ conclude that suppression of reproduction is more likely to occur under conditions of chronic stress and may involve actions at the level of the hypothalamus or pituitary. In addition, they indicate that there are likely to be species differences in the effect of glucocorticoids on gonadotropin secretion, and the presence of sex steroids and the sex of an individual are also likely to be factors.

Cytokines are members of the family of growth factors, and the interleukins in particular may act at the level of the hypothalamus where they may control the release of gonadotropin-releasing hormone, thus influencing the release of pituitary gonadotropins and ultimately gonadal steroid synthesis and release. Svechniko et al.⁶² have reported that testicular interleukin (IL)-1 may play a role in the paracrine regulation of Leydig cell steroidogenesis in rats. Others have reported that testicular interleukins may play an important functional role in both normal testicular function and under pathological conditions.⁶³ There is some evidence in the bovine that cytokines such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ may play a role in nitric oxide regulation in luteal endothelial cells by increasing inducible nitric oxide synthase (iNOS) activity, thereby accelerating luteolysis, while progesterone is thought to suppress iNOS expression in bovine luteal cells.⁶⁴ Whether these or other cytokines have similar effects on testicular cells of the bull has yet to be reported.

Steroid synthesis by Leydig cells

Since the beginning of the twentieth century, Leydig cells have been considered the probable source of testicular androgens.⁶⁵ Berthold⁶⁶ was the first to observe from experimentation with the rooster that the testes produced a substance that influenced secondary sex organ development and maintenance.²⁶ The first isolation of an androgen, androsterone, from human urine⁶⁷ and the crystallization of testosterone from bull testes¹¹ established the major site of testosterone production as the testis. Extensive literature has emerged over the past 50 years on the function of the Leydig cell. These studies, employing a variety of techniques, have identified and confirmed the Leydig cells as the primary source of testicular androgens and elucidated the important pathways in androgen biosynthesis.^{5, 26, 52, 68} LH has also been confirmed as the major pituitary hormonal stimulus on the Leydig cells.⁴⁶ Ewing et al.⁶⁸ have suggested that because Leydig cells are concentrated in clusters in the interstitial tissue of the testis, they must influence seminiferous tubular and peripheral androgen-dependent functions (accessory sex glands) by hormonal signals rather than by cell-to-cell interaction.

The primary steroids produced and secreted by the bull testis are shown in Table 2.1. The conversion of the precursor cholesterol to androgens and estrogen is facilitated by a series of enzymatic reactions involving hydroxylation, dehydrogenation, isomerization, C–C side-chain cleavage (lyase), and aromatase activity. Testosterone is now recognized as the principal steroid responsible for the endocrine functions of the testis since it is synthesized in copious amounts by mammalian Leydig cells. The Leydig cell has also been identified as a major source of other androgens and estrogens.^5,69 In addition, the Leydig cells of the boar testis produce large amounts of the musk-smelling steroids, Δ¹⁶-androstenes.⁷⁰

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