Barry T. Hinton

The Blood-Testis and Blood-Epididymis Barriers Are More Than Just Their Tight Junctions

The terms blood-testis barrier (BTB) or blood-epididymis barrier (BEB), are often described as Sertoli cell-Sertoli cell tight junctions (TJs) or TJs between the epithelial cells in the epididymis, respectively. However, in reality, the BTB and BEB are much more complex than just the TJ. The focus of this minireview is to remind readers that the complete BTB and BEB are comprised of three components: anatomical, physiological, and immunological. The TJs form the anatomical (physical) barrier that restricts passage of molecules and cells from entering or exiting the lumen. The physiological barrier is comprised of transporters that regulate movement of substances in or out of the lumen, thus creating a microenvironment, which is critical for the proper development and maturation of germ cells. The immunological barrier limits access by the immune system and sequesters the majority of the autoantigenic germ cells. Combined with the overall immune-privilege of the testis, this suppresses detrimental immune responses against the autoantigenic germ cells. These three components on their own do not create a complete functional barrier; instead, it is the interaction between all three components that create a barrier of maximal competence.

Cellular Signaling by Fibroblast Growth Factors (FGFs) and Their Receptors (FGFRs) in Male Reproduction

The major function of the reproductive system is to ensure the survival of the species by passing on hereditary traits from one generation to the next. This is accomplished through the production of gametes and the generation of hormones that function in the maturation and regulation of the reproductive system. It is well established that normal development and function of the male reproductive system is mediated by endocrine and paracrine signaling pathways. Fibroblast growth factors (FGFs), their receptors (FGFRs), and signaling cascades have been implicated in a diverse range of cellular processes including: proliferation, apoptosis, cell survival, chemotaxis, cell adhesion, motility, and differentiation. The maintenance and regulation of correct FGF signaling is evident from human and mouse genetic studies which demonstrate that mutations leading to disruption of FGF signaling cause a variety of developmental disorders including dominant skeletal diseases, infertility, and cancer. Over the course of this review, we will provide evidence for differential expression of FGFs/FGFRs in the testis, male germ cells, the epididymis, the seminal vesicle, and the prostate. We will show that this signaling cascade has an important role in sperm development and maturation. Furthermore, we will demonstrate that FGF/FGFR signaling is essential for normal epididymal function and prostate development. To this end, we will provide evidence for the involvement of the FGF signaling system in the regulation and maintenance of the male reproductive system.

2 The Role of the Epididymis in the Protection of Spermatozoa

Publisher Summary This chapter discusses the structure and function of the epididymis, including the formation of a luminal fluid microenvironment, sperm maturation, and sperm transport. It discusses specifically on the manner, by which the epididymis protects the maturing spermatozoa. Spermatozoa leave the testis neither fully motile nor able to recognize or fertilize an egg, but must traverse a long duct, the epididymis, to acquire these abilities. These transformations of the spermatozoa are collectively referred to as sperm maturation. Even though sperm have developed the ability for motility and to fertilize, they still need to undergo a further maturation step in the female reproductive tract before they achieve their full fertilization potential. In addition, to the numerous changes that the maturing spermatozoa are undergoing during their transit along the epididymal duct, the epididymis also protect spermatozoa from the external environment. The epididymis is an androgen-dependent organ, where many aspects of the epididyma1 function, including the processes of sperm maturation and storage require the presence of androgens.

CHAPTER 22 – The Epididymis

Throughout embryonic and early postnatal development, the mammalian epididymis changes from a straight tube to a highly coiled, complex duct that links the efferent ducts to the vas deferens. Overwhelming evidence points to the importance of this tissue in transforming spermatozoa leaving the testis as immotile cells, unable to fertilize oocytes, into fully mature cells that have the ability both to swim and to recognize and fertilize eggs. Under normal conditions, the acquisition of these functions is essentially completed by the time sperm enter the proximal cauda epididymidis. In addition to sperm maturation, the epididymis also plays an important role in sperm transport, concentration, protection, and storage. A highly specialized and region-specific microenvironment is created along the epididymal lumen by active secretion and absorption of water, ions, organic solutes, and proteins as well as by the blood–epididymis barrier. The primary factor regulating epididymal function is androgens, but there is mounting evidence that estrogens, retinoids, and other factors coming directly into the epididymis from the testis through the efferent ducts, such as growth factors, also play specific regulatory roles. Several epithelial cell types, each showing selective expression of genes and proteins, are differentially distributed along the duct; each cell type shows highly regionalized expression of a wide array of markers. Both epididymal epithelial cells and spermatozoa in the lumen are targets for xenobiotics; such exposures can result in undesirable toxic effects or may provide the basis for the development of novel male contraceptive agents. During aging, both the epididymal epithelium and the germ cells in the lumen undergo a series of dramatic changes. The explosion of knowledge we are witnessing regarding all aspects of epididymal structure and function is likely to lay the basis for a new fundamental understanding of epididymal cell biology and novel therapeutic approaches targeted at this organ.

Development and morphogenesis of the Wolffian/Epididymal Duct, More Twists and Turns

The epididymis serves a critical function of preparing the male germ cells for fertilization. In order for the epididymis to carry out this role it must undergo a highly coordinated succession of molecular and morphogenic events during development. These events begin with the formation of the Wolffian or nephric duct, the embryonic precursor of the male reproductive system, and end with the three-dimensional coiled postnatal epididymis that is comprised of several distinctly functional segments. How the duct changes from a simple straight tube to a highly convoluted structure will be the focus of this article. In reviewing the literatures current understanding of epididymal morphogenesis, we will highlight some of the classic morphological studies and discuss some of the more recent genetic models that have all served to contribute to our understanding of this system. Where published information is scarce we will provide potential hypotheses that warrant further investigation and may open up new directions of exploration using the epididymis as a model for tubular morphogenesis.

The Development of the Epididymis

The adult epididymis is a highly convoluted tubule that performs a variety of functions including sperm protection, maturation, concentration and storage. These functions can only be accomplished following a series of morphological changes that occur during development. These changes result in the development of a single tubule that differs in several regions with respect to morphology, function and gene expression. Therefore, it is not surprising that the development of the epididymis is a highly regulated event and is not yet completely understood. This chapter will attempt to summarize the events that take place leading to the development and differentiation of the adult epididymis. Major key studies will be summarized and important questions that remain unanswered will be addressed.

Organic Cation/Carnitine Transporter, OCTN2, Is Differentially Expressed in the Adult Rat Epididymis

Abstract l-Carnitine must be transported against a substantial concentration gradient across the epididymal epithelium to achieve high intraluminal levels, approximately 50 mM in the cauda. Recently, an organic cation transporter, OCTN2, was cloned from rat intestinal epithelium and shown to transport l-carnitine in a sodium-dependent manner. To test the hypothesis that OCTN2 was present in the epididymis, primers were designed based on the published OCTN2 mRNA sequence. A 1.9-kilobase OCTN2 cDNA from rat epididymis was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned. Northern analysis demonstrated the presence of OCTN2 transcripts in the epididymis, with highest expression in the distal caput and corpus. To localize the protein, an antibody raised against a carboxy-terminal peptide of OCTN2 was produced in rabbits and used for Western blot analysis and immunohistochemistry. The antibody recognized a band of approximately 65 kDa in Western blots using epididymal lysates. Immunohistochemical studies demonstrate that OCTN2 is present in the basolateral membrane of epithelial cells in the distal caput, corpus, and proximal cauda epididymides. In conclusion, OCTN2 is present in the rat epididymis in a region-dependent manner and is likely to be responsible for the transport of l-carnitine into the cells of the epididymal epithelium.

Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood–brain barrier

Carnitine β‐hydroxy‐γ‐(trimethylammonio)butyrate – a compound necessary in the peripheral tissues for a transfer of fatty acids for their oxidation within the cell, accumulates in the brain despite low β‐oxidation in this organ. In order to enter the brain, carnitine has to cross the blood–brain barrier formed by capillary endothelial cells which are in close interaction with astrocytes. Previous studies, demonstrating expression of mRNA coding two carnitine transporters – organic cation/carnitine transporter 2 (OCTN2) and B0,+ in endothelial cells, did not give any information on carnitine transporters polarity in endothelium. Therefore more detailed experiments were performed on expression and localization of a high affinity carnitine transporter OCTN2 in an in vitro model of the blood–brain barrier by real‐time PCR, western blot analysis, and immunocytochemistry. The amount of mRNA was comparable in endothelial cells and kidney, when referred to house‐keeping genes, it was, however, significantly lower in astrocytes. Polarity of OCTN2 localization was further studied in an in vitro model of the blood–brain barrier with use of anti‐OCTN2 antibodies. Z‐axis analysis of the confocal microscope pictures of endothelial cells, with anti‐P‐glycoprotein antibodies as the marker of apical membrane, showed OCTN2 localization at the basolateral membrane and in the cytoplasmic region in the vicinity of nuclei. Localization of OCTN2 suggest that carnitine can be also transported from the brain, playing an important role in removal of certain acyl esters.

Characterization of Fibroblast Growth Factor Receptors Expressed in Principal Cells in the Initial Segment of the Rat Epididymis

Abstract Studies from our laboratory support a model in which growth factors produced in the testis reach the epididymis via the luminal system and play an important role in maintaining the function of epithelial cells, particularly in the initial segment. Previous work showed that γ-glutamyl transpeptidase (GGT) mRNA IV, which is highly expressed in the rat initial segment, may be under the control of luminal fibroblast growth factor 2 (FGF-2) from the testis. The current studies were undertaken to identify which fibroblast growth factor receptors (FGFRs) are present in the principal cells of the rat initial segment and to identify other potential ligands for these receptors in rat rete testis fluid (RTF). Immunoblot analysis revealed that FGFRs 1–4 were present, and reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that both the IIIb and IIIc splice variants of FGFRs 1–3 were expressed. However, RT-PCR using RNA isolated from principal cells collected by laser capture microdissection revealed only FGFR-1 IIIc. Additional PCR analysis established that both the α and β forms of FGFR-1 IIIc were expressed in principal cells. Both FGF-4 and FGF-8 were present in rat RTF, as determined by immunoblotting. Thus, FGF-2, -4, and -8, found in RTF, may act upon FGFR-1 IIIc in the principal cells of the initial segment to regulate GGT mRNA IV expression.

5′ Hox Genes and Meis 1, a Hox-DNA Binding Cofactor, Are Expressed in the Adult Mouse Epididymis

Abstract Hox genes determine the formation of segmented structures during development. The epididymis shows a segmented organization in its structure and function beyond embryogenesis. This study examined the adult mouse epididymis and vas deferens for expression of 5′ hox genes and a hox-DNA binding cofactor. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the expression of hoxa-9, hoxa-10, hoxa-11, hoxd-9, and hoxd-10 in all regions including the vas deferens. Semiquantitative RT-PCR revealed highest mRNA levels for hoxa-11 in the distal part of the epididymis and vas deferens, and this was confirmed by Northern blot analysis. To determine protein presence an antibody raised against a peptide N-terminal to the homeodomain of hoxa-11 was produced in rabbits. The antibody recognized a band of approximately 37–39 kDa in Western blot analysis. Immunohistochemistry indicated the presence of hoxa-11 in the nuclei of the epithelial cells with some staining in the cytoplasm. Staining was also detected in nuclei of interstitial cells throughout the entire organ and the vas deferens. A DNA binding cofactor for hoxa-11, Meis 1, was investigated for its presence in the epididymis. Semiquantitative RT-PCR identified both transcripts for Meis 1 (Meis 1a and Meis 1b) in all regions. Protein presence was confirmed by Western blot analysis, and this detected one band of approximately 53–55 kDa. Immunohistochemistry localized Meis 1 in the nuclei of interstitial cells throughout the entire organ and the vas deferens. Our study provides preliminary data from which we suggest the involvement of homeodomain transcription factors in the maintenance of segmental function of the adult epididymis and vas deferens.