has yet to be clearly demonstrated in the male bovid, but with the advent of new technologies this may be possible [107]. However, Kohsaka et al. [108, 109] have demonstrated, utilizing immunohistochemical studies, the binding of relaxin to spermatozoa and suggested that this activity may impart physiological effects on sperm motility and fertility. Moreover, these authors suggested that relaxin activity in its relationship to sperm motility in domestic species may serve as a possible index for predicting the fertilizing ability of sires. Subsequent studies have demonstrated that relaxin improves sperm motility in a number of species, including the human male [110], boar [111], and bull [112]. Furthermore, others have demonstrated that use of relaxin treatment of seminal plasma induces sperm capacitation and acrosome reaction in boar [113] and domestic bull [114] spermatozoa. Other studies have shown that relaxin treatment improved the fertilizing capabilities of fresh boar spermatozoa [115] and fresh or frozen–thawed spermatozoa of the buffalo bull [116, 117] and stallion [118]. Thus, it is evident from these studies that relaxin may play an important role in sperm motility and function. While the RLN1 gene may be lost in the bovid, the physiological benefits of relaxin lie in its potential use as a semen supplement when using either fresh or frozen–thawed bull spermatozoa to facilitate sperm motility and enhance fertilizing capacity during artificial insemination.
Seminiferous Tubules, Sertoli Cells, and Germinal Tissue
Within segments of the testicular parenchyma lie the seminiferous tubules, originally formed from the primary sex cords. The seminiferous tubules contain the Sertoli cells, also known as the nurse cells of the developing germinal cells, the spermatogonia, which are elongated cells found within the tubules. The Sertoli cells were first described by the Italian physiologist Enrico Sertoli in the early 1860s. Structurally, these cells are very important in that they form the blood–testis barrier, which separates the interstitial blood compartment of the testes from the adluminal compartment of the seminiferous tubules. In addition to Sertoli cells nurturing the development and maturation of the germinal cells through the various stages of spermatogenesis, these cells act as phagocytes by removing or consuming residual cytoplasm and surplus spermatozoa material. Sertoli cells, which are also very active secretory cells, produce anti‐Müllerian hormone (AMH), a glycoprotein secreted during early fetal life by the testis to direct the appropriate development of the male reproductive tract by causing the regression of the Müllerian ducts [119, 120]. Mutations of the AMH gene lead to persistent Müllerian duct syndrome in male human patients [121]; whether the same observation is true in cattle has not been reported. However, recent studies successfully employed the use of AMH profiles as a novel biomarker to evaluate the existence of functional cryptorchid testis in Japanese black calves [122]. A more comprehensive review of Sertoli cell cytology may be found in the review by de Kretser and Kerr [33].
In addition, Sertoli cells secrete two closely related protein complexes of the transforming growth factor cytokine superfamily (activin and inhibin) that have opposite regulatory effects on the synthesis and regulation of pituitary FSH secretion. Activin upregulates while inhibin downregulates FSH synthesis and secretion [123, 124]. Both of these hormones are secreted after puberty and play an important role in mammalian reproduction, which is coordinated by assorted neural, neuroendocrine, endocrine, and paracrine cell–cell communication pathways [125]. Activin is also thought to facilitate androgen synthesis in the testis by enhancing LH, thereby promoting spermatogenesis. In male mammals, inhibin production is thought to be promoted by testicular androgens and may function locally in the testis to help regulate spermatogenesis. Much less is known about inhibin in relation to its mechanism of action, but it is thought that it may compete with activin for the binding of the activin receptor and/or binding to the inhibin‐specific receptor [124]. However, Phillips [126] in reviewing the literature notes that inhibin not only acts as a feedback regulator of FSH in the male, but also may be an important paracrine and autocrine regulator of testis function. It has been shown from molecular studies that both forms of inhibin (A and B) are produced by the bovine testis and that the proportion of the mature forms of inhibin A and inhibin B increases as bulls age, but that total inhibin production by Sertoli cells decreases [127]. In a later study, Kaneko et al. [128] reported that inhibin A and inhibin B increased in the testis of bulls during postnatal development, and that immunoreactive inhibin A could be detected in the plasma of these bulls. Others have attempted to use blood concentrations of inhibin and gene expression along with other endocrine and genetic markers as a predictor of fertility in Brahman bulls [129]. Unfortunately, there appears to be little information in the current literature on the role activin plays in the bovine male. Its role as a paracrine regulator in the testis is presumed but has not been conclusively demonstrated in domestic species, although in vitro studies in porcine Leydig cells would suggest a role in androgen synthesis [126]. However, it is thought to be an important intrafollicular factor in the regulation of follicle selection in the bovine female [130, 131]. A role in inflammatory processes for activin has been postulated [126].
Additional factors secreted by the Sertoli cell include ABP, estradiol (by conversion of testosterone via the aromatase enzyme complex), glial cell‐derived neurotrophic factor (GDNF), transferrin [132], and the more recently identified ERM transcription factor. Testicular transferrin and ABP are two glycoproteins produced and secreted into the lumen of the tubule by Sertoli cells, and which have important biological effects on differentiation and maturity of sperm [133, 134]. ABP is a glycoprotein that binds specifically to testosterone, DHT, and 17β‐estradiol. By binding to ABP, the steroid hormones testosterone and DHT are less lipophilic and become more concentrated within the lumen of the seminiferous tubules, thereby helping to facilitate spermatogenesis. ABP synthesis is regulated by FSH activity on the Sertoli cells, which may be enhanced by insulin and testosterone. ABP can especially combine with testosterone to maintain a high concentration of androgen in the seminiferous tubule, which facilitates the development and maturation of spermatogenic cells. Androgen is transferred to the target cells and via the nuclear transfer mechanism into the nucleus to combine with androgen receptors, which regulate translation of the target genes. The characteristic of ABP combining with androgen shows that ABP participates in sexual differentiation and maturation of germ cells [132].
Sertoli cell gene products include growth factors, metabolic enzymes, transport proteins (e.g. transferrin, ABP), inhibin, proteases, antiproteases, energy metabolites, and structural components [135]. Transferrin is a major secretory product of differentiated Sertoli cells and is postulated to transport iron sequestered by the blood–testis barrier to the developing germ cells. Fe3+ in blood is transferred to Sertoli cells, combined with transferrin synthesized by Sertoli cells, and further transferred to spermatogenic cells at the development stage to promote germ cell growth and maturation. It is well known that the synthesis and secretion of transferrin and ABP are regulated by FSH and androgen. The gonadotropin FSH is an important endocrine hormone required for the regulation of Sertoli cell function [136]. FSH has been shown to regulate the expression of most Sertoli cell genes, including the FSH receptor, ABP, transferrin, plasminogen activator, and aromatase [137–140].
GDNF is a small protein normally associated with the promotion of neuron survival [141]. Its role in testicular function is not fully understood, but there is some evidence to suggest that it may be involved in promoting undifferentiating spermatogonia, which ensures stem cell self‐renewal during the perinatal period [142]. Moreover, Johnston et al. [143] demonstrated in the adult rat that cyclic changes in GDNF expression by Sertoli cells are responsible for the stage‐specific replication and differentiation of stem spermatogonia, the foundational cells of spermatogenesis. Recent reports in the literature have demonstrated that GDNF, rather than induce proliferation of spermatogonia stem cells, enhances self‐renewal and increases survival rate of bovine spermatogonia in culture [144]. An interesting observation reported by Harikae et al. [145] is a rare case of a freemartin calf exhibiting the transdifferentiation of ovarian somatic cells into testicular somatic cells including Sertoli cells, Leydig cells, and peritubular myoid cells, and that the Sertoli cells stained positive for GDNF protein. They speculate that these observations suggest that mammalian XX ovaries may have a high potential for sexual plasticity. For a more comprehensive review of the function and regulation of Sertoli cells, the reader is referred to the excellent review by Russell and Griswold [146].
Overview