Endocrine and Exocrine Function of the Bovine Testes

Chapter 2
Endocrine and Exocrine Function of the Bovine Testes

Peter L. Ryan

College of Veterinary Medicine, Mississippi State University, Starkville, Mississippi, USA


The normal bovine male reproductive system consists of paired testes retained within a sac or purse-like structure known as the scrotum, which is formed from the outpouching of skin from the abdomen and consists of complex layers of tissue. The testes are accompanied by a number of supporting structures including spermatic cords, accessory sex glands (prostate, bulbourethral, paired vesicular glands), penis, prepuce, and the male ductal system. The testicular duct system is extensive and comprises the vas efferentia found within the testes, the epididymis, vas deferens, and the urethra, all of which are located external to the testes. The reader is referred to the excellent chapter on the anatomy of the reproductive system of the bull in this book (Chapter 1). The primary functions of the testes are to produce male gametes (spermatozoa) and the endocrine factors, such as steroid (testosterone) and protein hormones (inhibin, insulin-like peptide 3), that help regulate reproductive function of the bull in concert with hormonal secretions from the hypothalamus (gonadotropin-releasing hormone) and pituitary glands (luteinizing hormone, follicle-stimulating hormone). The testes consist of parenchymal tissue that supports the interstitial tissue and includes the steroid-producing Leydig cells, vascular and lymphatic system, and the seminiferous tubules within which the germinal tissue develops with the support of the nurse cells more commonly known as Sertoli cells. Chapter 4 discusses in detail the endocrine factors responsible for testicular development and initiation of spermatogenesis in the bull, and thus this chapter focuses more on the regulation and function of the adult testes. This chapter will not undertake a treatise of those conditions that disrupt testicular function but rather will focus, as practically as is possible, on what is known of the endocrine and exocrine function of the bovine testes. Much of the endocrine and exocrine function of the testes is similar across mammalian species, and where specific information is absent for the bovine, examples will be given from other domestic species when possible. It has not been possible to cite the many significant contributions to the field of endocrine and exocrine function of the testes. Thus, where and when possible, the reader is referred to selected citations for additional reading.

Historical perspective

It has been evident for many centuries that the testes exercise control over the characteristics of the male body. The results of castration in domestic animals and human males made this very clear, but provided no clues as to the mechanism of control. Pritchard1 noted from Assyrian records dating some 15 centuries bc that the castration of men was used as punishment for sexual offenders, which suggests that the effect of castration on fertility and behavior was recognized at that time. Knowledge of the effects of castration of livestock dates back to the Neolithic Age (c. 7000 bc) when animals were first thought to have been domesticated.2 The effects of castration were understood by Aristotle (300 bc) who provided very detailed and clear descriptions of testicular anatomy and function.3 It was not until the seventeenth century that a detailed account of testicular and penile anatomy was presented by Regnier de Graaf4 in a treatise on the male reproductive organs. De Graaf indicated the existence of the seminiferous tubules and suggested that the production of the fertile portion of the semen occurred in the testes. The first microscopic examination of the testes was undertaken by Antonie van Leeuwenhoek in 1667 where he demonstrated and reported the presence of germ cells in the seminal fluid.5

Detailed study of the testis began in the mid-nineteenth century. In 1840, Albert von Köllicker discovered that spermatozoa develop from cells residing in the testicular (seminiferous) tubules. This major discovery was followed by Franz Leydig’s6 description of the microscopic characteristics of the interstitial cells. Later, Enrico Sertoli,7 an Italian scientist, correctly described the columnar cells running from the basement membrane to the lumen of the tubuli seminiferi contorti (seminiferous tubules) of the testes, and Anton von Ebner is credited with introducing the concept of the symbiotic relationship between Sertoli cells and the developing germinal cells.8,9

The first clear demonstration that the testes are involved in an endocrine role was made by Arnold Berthold in 1849, while studying the testes of the rooster. He concluded that the regulation of male characteristics was brought about by way of blood-borne factors. The most compelling evidence for an endocrine function of the testes being associated with Leydig cells was presented by two French scientists, Bouin and Ancel in 1903. They reported that ligation of the vas deferens in dogs, rabbits, and guinea pigs was followed by degeneration of the seminiferous tubules, but no castration effects were observed and no degenerative changes of the interstitial cells, and thus concluded that internal secretions of the testes were synthesized by the Leydig cells.10 By the mid 1930s it was clear that the male hormone emanating from the testes was testosterone11 and that the function of the testes was controlled by pituitary hormones.12,13 Smith12 demonstrated that the pituitary gland must secrete substances (now known as gonadotropins) responsible for the stimulation of testicular growth and maintenance of function in the rat. Greep and Fevold14 restored Leydig cell function in hypophysectomized rats with crude preparations of luteinizing hormone (LH) and reestablished the male secondary sex characteristics. Further evidence was elucidated in favor of a steroid secretory function for Leydig cells from a study on postnatal development in bulls where changes in testicular androgen levels paralleled the differentiation of these cells.15 Using cell culture techniques, Steinberger et al.16 provided direct evidence that the Leydig cells are the primary source of steroid hormone synthesis in the testes. Later, it became apparent that the Leydig cells are essential for providing the androgenic stimuli that are required for the maintenance of spermatogenesis in the germinal epithelium.16, 17

The testis

The bovine testes are paired, capsulated, ovoid-like structures located in the inguinal region and suspended in a pendulous scrotum away from the abdominal wall. The proximal relationship of the testes to the abdominal wall varies and may depend on season and ambient temperatures. The cremaster muscle plays an important role in thermal regulation of the testis. The size of the testis varies with breed, but typically the adult testis weighs 300–400 g and is about 10–13 cm long and 5–6.5 cm wide.18 The tough fibrous capsule covering each testis consists of three tissue layers: the outer layer, the tunica vaginalis; the tunica albuginea, which consists of connective tissue composed of fibroblasts and collagen bundles; and the inner layer, the tunica vaginalis, which supports the vascular and lymphatic systems.19 The capsule is the main structure that supports the testicular parenchyma, the functional layer of the testes, which consists of the interstitial tissue and seminiferous tubules. The interstitial tissue is found in the spaces between the seminiferous tubules and consists of clusters of Leydig cells, which are primarily responsible for steroid hormone biosynthesis and secretion, along with vascular and lymph vessels that supply the testicular parenchyma. The seminiferous tubules originate from the primary sex cords and contain the germinal tissue (spermatogonia, the male germ cell) and a population of specialized cells, the Sertoli cells, which not only support the production of spermatozoa but also form tight junctions with each other, creating one of the most important components of the blood–testis barrier.20 This structure prevents the entry of most large molecules and foreign material into the seminiferous tubules that may disrupt normal spermatogenesis. The most important substances synthesized by the testes and released into the vascular system are peptide and steroid hormones. However, fluids from the seminiferous tubules may pass into the interstitial tissue via the basal lamina, where they may enter the testicular lymphatic and vascular systems, or into the tubule lumen via the apical surface of the Sertoli cells.19

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.


Figure 2.1 Bright-field and thermal images of 2-year-old Hereford bull testes. The images were taken using an FLIR SC600 thermography unit (FLIR Systems Sweden, AB) on June 25, 2013 at midday with atmospheric temperature of 29°C and humidity of 75%. (a) Bright-field image of the testes. (b) Thermal image of the testes as seen in (a), identifying three regions of interest (RO1, RO2 and RO3) as indicated by the vertical bars along which temperature values were obtained by means of the software ThermaCAM Research Pro 2.7. The image is pseudo-colorized with temperature scale bar to help visualize the change in temperature gradient from scrotal neck to scrotal tip. (c) Temperature values, minimum (Min), maximum (Max), the difference between Min and Max and the average (Avg) temperature for each region of interest (RO), and the standard deviation (st dev) which assess the variation of temperature in each region. Note the almost 4°C change in average temperature from RO1 to RO3.

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

Endocrine function of the testis

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 (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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