Richard Bronson, MD, Department of Obstetrics & Gynecology, Stony Brook University Medical Center Stony Brook, NY 11794-8091, USA. E-mail: Richard.Bronson@Stonybrook.edu
Citation Bronson R. Biology of the male reproductive tract: Its cellular and morphological considerations. Am J Reprod Immunol 2011; 65: 212–219
For many years, the focus of attention in the study of semen has been on spermatozoa, its major cellular component, given their importance in the process of reproduction, and the role of the seminal fluid as their transport medium. More recently, evidence has accumulated of the complexity of seminal fluid, its components that perturb the female reproductive tract in ways promoting both survival of spermatozoa there-in and facilitating the implantation of embryos within the endometrium, hence initiating pregnancy. These same factors, however, may also make the female reproductive tract susceptible to invasion not only by spermatozoa but viruses, playing a significant role in the male-to-female transmission of HIV. Knowledge of the histology, anatomy, and immunology of the male reproductive tract is essential in understanding its role in HIV pathogenesis.
The objectives of this short review are to allow the reader to become familiar with the anatomy and histology of the testes, to survey those immune-modulatory factors in semen that may prevent sensitization to sperm in women and promote embryo implantation, and to review the role of Sertoli cells in the formation of the blood–testes barrier (BTB), in the context of preventing autoimmunity to sperm. I pose two immunologic puzzles that could shed light on the male-to-female sexual transmission of HIV. As sperm are foreign cells that enter the female reproductive tract at coitus, why is an immune response against them not mounted, as it is against microbes such as chlamydia and yeast? Why don’t most men develop autoimmunity to their own sperm and subsequent orchitis, as spermatogenesis begins at puberty, at a time after which the immune system recognizes the body’s own antigens as ‘self’?
The bulk of the volume of the testes consists of the seminiferous tubules within which spermatozoa are produced. These connect through the rete testis to the head of the epididymis and subsequently, to the vas deferens. The volume of the testes, palpated clinically, then correlates with the functional activity of spermatogenesis, increasing with puberty. Conversely, in those clinical conditions, in which spermatogenesis is severely impaired, such as Klinefelter’s syndrome, testes volume tends to be smaller than normal.1
The process of sperm formation can be divided into three separate components:
• Spermatogenesis – the formation of sperm cells that have undergone first and second meiotic divisions, but have remained round in shape.2
• Spermiogenesis – during which sperm undergo a morphologic change in shape from round cells to potentially motile, tadpole-shaped cells.3,4
• Spermiation – the release of spermatids into the seminiferous tubule lumen from their relationship with Sertoli cells.5
The entire process of sperm production occurs over approximately 10 weeks.5
Spermatozoa leaving the testes and entering the epididymis do not possess the ability to fertilize eggs, but acquire this ability during their transit through the epididymis. This process is not yet completely understood, but is associated with acquisition of propulsive motility and alterations in the sperm plasma membrane and glycocalyx.6,7 Only approximately 1 cc of the ejaculate volume (normal range 2–6 cc) is made up of sperm-containing fluid of the vas deferens. The remaining ejaculate volume reflects contributions of the male accessory glands (the prostate and seminal vesicles). The latter secretions contain prostaglandins and TGF-beta, which play potential roles in immunosuppression and in sperm transport within the female reproductive tract.
If one examines the histology of the testes on cross-section, the seminiferous tubule will be seen to be surrounded by a layer of myoid cells on which the spermatogonia rest, the progenitor cells from which spermatocytes undergoing meiosis are produced.2 Sertoli cells ascend from the base of the seminiferous tubule toward its lumen, like ‘trees of a forest.’ They play roles in the endocrine regulation of the pituitary gonadotropins, as well as in the segregation of spermatids & spermatocytes from the systemic immune system, and in the process of spermiogenesis.4 The interstitial compartment located between the seminiferous tubules contains Leydig cells as well as lymphocytes and blood vessels. Leydig cells synthesize testosterone and estradiol under the stimulus of luteinizing hormone (LH) secreted by the pituitary, which is regulated through negative feedback at the level of the pituitary and hypothalamus.1 Inhibin produced primarily by the Sertoli cells feeds back to the anterior pituitary in a negative manner, regulating the secretion of follicle-stimulating hormone (FSH).1,8
Primary spermatocytes originating from the spermatogonia ascend toward the tubular lumen, supported by Sertoli cells. Their architecture within the seminiferous tubules is best seen in the condition known as Sertoli cell only syndrome, in which spermatogonia are absent, associated with azoospermia. During spermiogenesis, round spermatids undergo a loss of cytoplasm, the formation of sperm tail that allows cell motility and a mid-piece containing mitochondria that provide energy for sperm motility. The acrosome is also created during this process. This structure located over the rostral portion of the spermatozoon head is essential for successful fertilization.9 Sperm are then cast off into the seminiferous tubal lumen (spermiation). Dynamic endoplasmic specializations at the base and apex of Sertoli cells play active roles in the creation of an adluminal compartment isolated from the immune system, in the ascent of maturing germ cells with the seminiferous tubule, and their release into the tubular lumen10 (Fig. 1).
The acrosome is a specialized granule, which contains a trypsin-like enzyme (acrosyn),11 the multi-functional adhesion molecule vitronectin,12 and other as yet not well-known moieties that play roles in gamete interactions that lead to fertilization.13 Sperm must first undergo a process termed capacitation within the female reproductive tract that endows them with the ability to fertilize, allowing the sperm to acrosome react.9 This process involves alteration in the sperm glycocalyx as well as loss of plasma membrane cholesterol.14 The ‘acrosome reaction’ occurs when sperm bind to a glycoprotein of the zona pellucida that surrounds the unfertilized egg.15 Following zona binding, sperm receptors are cross-linked, leading to an increase in intracellular calcium and the promotion of the ‘acrosome reaction.’16 During this process, the sperm plasma membrane fuses with the outer acrosomal membrane, creating fenestrations through which acrosomal contents are released.9 The sperm plasma membrane and outer acrosomal membrane are lost from the rostral portion of the sperm head, and the completely acrosome-reacted sperm (now bounded by the inner acrosomal membrane) penetrates through the zona pellucida, entering the perivitelline space, and subsequently adhering to the egg surface, the oolemma.17 The egg recognizes this adherence in an as yet undefined manner, possibly through specific receptor-ligand interactions, and subsequently plays an active role in incorporating the sperm within its cortical ooplasm.18 At this time, the egg undergoes activation, with the completion of the second meiotic division, release of the second polar body, and release of cortical granules in the perivitelline space, which alter the zona pellucida, preventing the binding and penetration of secondary sperm.19
Evidence that Sertoli cells play a role in the morphologic changes sperm undergo during spermiogenesis has been provided in a series of experiments in mice, in which the adhesion molecule nectin-2 was knocked out.20 Males lacking nectin-2 expression are infertile, associated with alterations in the shape of their spermatozoa. These sperm exhibit altered motility as well and an impairment in their ability to adhere to both the zona pellucida and to the oolemma proper in vitro, associated with impaired fertilization. Alteration in the sperm tail beating was noted, and fewer sperm were found within the oviducts of wild-type females mated with nectin-2 knockout males than wild-type males. Subsequent studies have shown that nectin-2 is expressed by Sertoli cells and nectin-3, its counter receptor, is present on spermatozoa.21 Knockout of either of these molecules is associated with alteration of sperm shape, motility, and male fertility.
During sexual relations, semen is deposited in the vagina after ejaculation. Although the vaginal pH is approximately 4.5, due to the production of lactic acid by resident lactobacilli, during female sexual excitement, the vaginal pH rises toward neutral. Seminal fluid is slightly alkaline (pH 7.2 - 7.8) and has significant buffering capacity.22 In addition, the normal pH of cervical mucus in the absence of semen is approximately 7.0, in the late follicular phase of the menstrual cycle. The characteristics of cervical mucus change at this time, allowing the entry of spermatozoa into the uterus and Fallopian tubes. Recent studies by Ceballo et al.23 suggest that HIV binds to human spermatozoa via heparin sulfate on the sperm surface, most likely involving syndecans 3 and 4, rather a mannose receptor. In addition, they showed that spermatozoa were internalized and promoted the uptake of HIV by DC in culture, which subsequently exhibited a marked increase in the expression of HLA-DR, CD40, CD83, and CCR7. The authors speculated that spermatozoa transmit the virus to mucosal DC’s within the reproductive tract and might alter the immune response against HIV by modulating their function.
Two immunologic puzzles that may provide clues to the pathogenesis of HIV infection in women
• As sperm are foreign cells that enter the female reproductive tract at coitus, why is an immune response against them not mounted, as it is against microbes such as chlamydia and yeast.22,23
• Spermatogenesis begins at puberty, at a time after which the immune system recognizes the body’s own antigens as ‘self’. Why then don’t most men develop autoimmunity to their own sperm and subsequent orchitis?
Why don’t women develop an immunity to sperm?
The female reproductive tract is capable of mounting an immune response to pathogens.24,25 There is increasing evidence that seminal plasma, which had conventionally been viewed solely as a transport medium for sperm, plays additional roles beyond this within the female reproductive tract (Table I). Seminal plasma has potent immunosuppressive activity, which can principally be attributed to its high content of TGF-beta26,27 and PGE prostaglandins.28 Emami et al.29 have provided evidence for the involvement of members of the seminal kallikrein-related peptidase (KLK) cascade in activation of latent TGF-beta in seminal plasma. Skibinski et al30 have shown that seminal plasma inhibits the function of both NK cell and T lymphocytes, and that the E series prostaglandins are responsible for the major portion of this suppression. Other factors in semen that may prevent immunity to sperm include the sperm themselves,31 prostasomes32,33 polymines,34 cytokines,35–37 soluble Fc-gamma receptors,38 pregnancy-associated plasma protein A (PAPP-A),39 vascular endothelial growth factor (VEGF),40,41 and seminal T cells.42
Table I. Semen-derived Factors That Modulate the Immune Response
Soluble Fc-gamma receptors
The concentration of PGE prostaglandins in human semen is many times higher than in other areas of the body, and semen contains 19-hydroxy PGE, which is not found elsewhere. The effects of the seminal prostaglandins are two-fold.28,30 First, a cAMP-mediated effect on T cells inhibiting clonal proliferation, as well as natural killer cell function, and biasing CD4 cells to T-helper-2 pattern of cytokine production away from one favoring a cell-mediated response. Second, PGE is a potent agent inducing a type 2 phenotype in dendritic cells, through its capacity to inhibit IL-12. Hence, at the level of the antigen-presenting cell, PGE and 19-hydroxy PGE alter the balance of cytokines, stimulating IL-10 and inhibiting IL-12 released by these cells, reinforcing its direct effects and inducing tolerance of antigens that are presented together with the IL-10. While necessary for the survival of the spermatozoa, such tolerance may have adverse effects, in the face of infection. Viruses which can be transmitted in semen (such as HIV & HPV) and other invading organisms would benefit from this switch in cytokines and the inhibition of the cell-mediated defenses. Not only is the initial immune response affected, but repeated exposure to semen could diminish immune surveillance and the removal of virally infected cells.
TGF-beta is now known to be a principal mediator of oral tolerance.26,27 The seminal vesicle is the principal source of TGFβ in rodents, where its synthesis is regulated by testosterone. In contrast, the prostate has been identified as a major site of TGF-beta in men.43 The seminal fluid content of TGF-beta is high, approximately five-fold that of serum and similar to that of colostrum. The normal range for TGF-beta in fertile men has been shown to be approximately 40–150 ng/mL, which remains relatively constant over time. Upon deposition in the female reproductive tract at coitus, seminal TGF-beta interacts with uterine and cervical epithelial cells, to initiate a cascade of downstream effects.44,45 It has been shown to be a principal agent in the post-coital inflammatory response, in mice, resulting in the recruitment and activation of leukocytes, including neutrophils, macrophages, and dendritic cells. Epithelial cells up-regulate expression of several pro-inflammatory cytokines and chemokines within several hours of coitus. In humans, exposure to semen induces neutrophil recruitment into the superficial epithelial layers of the cervix.
In addition to preventing aberrant immunity to spermatozoa, seminal fluid components derived from the seminal vesicles have been implicated in inducing an immune response that promotes embryo implantation. Robertson et al. have provided evidence, in mice, that exposure to seminal fluid TGF-beta plays a role in promoting the implantation of embryos within the uterus.43 TGF-beta derived from the seminal vesicle binds to epithelial cells within the uterus, altering their local secretion of cytokines. Fetal loss and abnormalities are considerably greater when embryos are transferred to recipients after pseudopregnancy is achieved when female mice are mated with seminal-vesicle-deficient males without exposure to male seminal fluids, compared with intact males. Preliminary evidence suggests a role for seminal fluid-derived factors in promoting embryo implantation in humans, although the clinical results are inconsistent. Gutsche et al.45 studied the influence of seminal plasma on the mRNA expression of cytokines in human endometrial epithelial and stromal cells in culture, demonstrating a concentration-dependent stimulation of IL-1 beta, Il-6, and LIF mRNA expression. Kimura et al.46 analyzed endometrial NK cells for their expression of CD16 and CD56 by flow cytometry, providing preliminary evidence that seminal plasma exposure recruited CD56 (bright) NK cells into the endometrium. Clinical studies performed at the time of laboratory-assisted reproduction have been inconsistent. Billinge et al. found that embryo implantation rates were higher in women exposed to raw semen at the time of follicular aspiration, during in vitro fertilization and embryo transfer, than in its absence.47 This phenomenon was observed in a subpopulation of women with occluded fallopian tubes, eliminating the possibility of in vivo fertilization of oocytes that may not have been retrieved at follicular aspiration. Subsequently, inconsistent results were obtained following deposition of seminal fluid intravaginally during IVF-ET. Fishel and associates failed to observe a difference in pregnancy rates when semen was deposited intravaginally, immediately after the time of oocyte recovery.48 Tremellen et al. observed no difference in pregnancy rates following transfer of frozen embryos, in a group of women who had coitus at the time of embryo transfer versus a sexually abstinent group, but the proportion of viable pregnancies at 6 weeks’ gestation was higher in the former group (odds ratio 1.48, P = 0.036).49 In another study, when cryopreserved seminal plasma was placed intravaginally just after follicular aspiration, the clinical pregnancy rate was 37.3% in the SP group versus 25.7% in the saline control group, but this difference did not reach statistical significance.50 Embryo implantation rates were not different in a third study in couple who had coitus at least once 12 hr after embryo transfer.51 A study in which seminal fluid was placed intravaginally at the time of intrauterine insemination (IUI) with spermatozoa washed out of semen revealed no difference in pregnancy rate when compared with a saline control.52 Unfortunately, all of these studies were of small size and did not define their clinical populations well.
Why don’t men develop autoimmunity to sperm?
Prevention against an autoimmune response to testicular autoantigens occurs through two independent mechanisms: (i) The confinement of most sperm-associated antigens by a strong but incomplete tissue barrier. (ii) Dynamic and less well-defined tolerance mechanisms that control autoreactive lymphocytes. More than 40 years ago, Johnson & Setchel cannulated the rete testes of rams and collected testicular fluid for analysis, noting the very low concentrations of proteins, including immunoglobulins, when compared with serum and lymph.53 They proposed the presence of a blood–testis permeability barrier around the seminiferous tubules. Initially, this was felt to be at the level of the myoid cells surrounding the base of the tubule. Subsequently, Dym & Fawcett54 studied the tight junctions in the seminiferous epithelium and the peritubular contractile layer at high magnification in rats, investigating the permeability of these junctions to lanthanum nitrate, a very small electron-opaque tracer used in testing the patency of intracellular clefts. They demonstrated that the blood–testis barrier existed at the level of tight junctions between Sertoli cells, what created a basal compartment containing lanthanum, separated from an adluminal compartment that did not.
Cell surface interactions in the seminiferous epithelium are very dynamic. This epithelium consists of columnar Sertoli cells, along whose surface different generations of germ cells progress toward the tubular lumen while undergoing spermatogenic differentiation.3,4 The Sertoli cell lateral membrane is involved in dynamic contact with the germ cells, as well as in connecting adjacent Sertoli cells to each other by a belt of occluding junctions that provide structural integrity to the BTB. The domains of the Sertoli cell involved in anchoring late spermatids as well as those involved in inter-Sertoli junctional contacts in which the Sertoli cell membrane is paralleled by a thick bundle of actin filaments, the so-called ectoplasmic specialization (ES).4,10
The BTB physically divides the seminiferous epithelium into basal and apical (or adluminal) compartments. Besides its function as an immunologic barrier to segregate post-meiotic germ cell antigens from the systemic circulation, it creates a microenvironment for germ cell development and confers cell polarity. During spermatogenesis, the BTB must physically disassemble permitting the passage of preleptotene and leptotene spermatocytes. Studies have shown that this dynamic process is regulated by transforming growth factor-beta 3 (TGF-beta-3) and tumor necrosis factor-alpha.55
Under what conditions do men develop autoimmunity to sperm?
Antisperm antibodies (ASA) are detected in approximately 70% of men who have undergone vasectomy.56 These antibodies have also been associated with obstructive azoospermia secondary to cystic fibrosis and with unilateral or bilateral congenital absence of the vans deferens.57 Autoimmunity to sperm can also occur following testicular trauma or following mumps orchitis, which may occur in post-pubertal men but is rare before puberty. In addition, approximately 5% of men from infertile couples have been found to have autoimmunity to sperm, associated with a negative history for other etiologies (Table II).58
Table II. Conditions Associated With Autoimmunity to Spermatozoa
Congenital Absence of the Vas Deferens
Post-pubertal mumps orchitis
Following vasectomy reversal, pregnancy rates are reduced when these ASA are present in the seminal fluid or detected on spermatozoa. However, this occurs relatively infrequently when men who have had vasectomy reversal are studied. Meinertz and colleagues studied a group of 216 men following vasovasostomy with mixed antiglobulin reaction (MAR) for IgG, IgA, and IgA secretory antibodies bound to sperm. ASA in serum and seminal plasma were detected by agglutination tests.59 In the subgroup with a pure IgG response, the conception rate reached 85.7%, whereas only 42.9% of men who also had IgA on their sperm achieved a pregnancy. When 100% of the spermatozoa were coated with IgA, the conception rate was reduced to 21.7%.
Isahakia et al.60 have shown, in baboons, that new antigens are expressed on developing spermatocytes and spermatids after initiation of spermatogenesis. Three monoclonal antibodies (Mabs) raised in mice immunized with baboon sperm were used to study the stage-specific expression of sperm-associated antigens on intratesticular sperm. One of these Mab’s recognized a moiety on the sperm tail and the other over the anterior acrosomal region of the sperm. The tail antigen was absent in 2- and 3-year-old baboon testes, first appearing in spermatids located close to the lumen of the seminiferous tubules at about 4 years of age. The acrosomal antigen was recognized in late pachytene spermatocytes and round spermatids in a 3-year-old animal, but failed to be demonstrated in a 2-year-old juvenile baboon.
These antigens, to which the immune system may not be tolerant, could play a role in the genesis of autoimmunity sperm. As men with acquired sperm obstruction (secondary to vasectomy) develop autoimmunity to sperm, we asked whether men with cystic fibrosis, the majority of whom exhibit obstructive azoospermia due to congenital absence of the body & tail of the epididymis, the vas deferens, and seminal vesicles, exhibited ASA in their serum. We also wanted to determine whether there was a relationship between puberty (at which time spermatogenesis becomes active) and the development of autoimmunity to sperm. We studied 15 males, using an Immunobead binding assay, to detect the presence of ASA in their serum.61 Six of 7 post-pubertal males (ages 18-33) were found to possess ASA in their serum. These men were judged post-pubertal by their testes volume and serum testosterone levels. Conversely, none of 8 pre-pubertal (ages 9–11) were found to have autoimmunity to sperm. An additional control consisted of 16 diabetic post-pubertal males, one of whom was found to exhibit ASA.
There is increasing evidence that the blood–testes barrier in itself is not sufficient to prevent autoimmunity to sperm. Immunogenic autoantigens have been detected in mice on preleptotine spermatocytes, which were located outside of a blood–testes barrier within the seminiferous tubules.62 Evidence for active regional regulation against an autoimmune response to these antigens has been obtained in the study of mice that have undergone thymectomy within 3 days of birth.63 In certain strains, neonatal thymectomy leads to the development of orchitis. Regulatory T lymphocytes have been identified within the interstitium of the testes in these animals,64 and autoimmune orchitis can be prevented by infusion of normal T cells. T cells are also present within seminal fluid and gain entry of the female reproductive tract at coitus.42 It has been speculated that these cells could play roles in altering the female reproductive tract response to spermatozoa. These same cell-medicated immune perturbations might play roles in the pathogenesis of HIV transmission.
Evidence has accumulated of the complexity of seminal fluid, its components that perturb the female reproductive tract, altering its ability to mount an immune response against spermatozoa (foreign invading cells of another individual), and facilitating the implantation of embryos within the endometrium. These same factors that promote the establishment of pregnancy, however, may also make the female reproductive tract susceptible to invasion not only by spermatozoa but viruses, playing a significant role in the male-to-female transmission of HIV. An understanding of the histology, anatomy, and immunology of the male reproductive tract is essential in understanding its role in the pathogenesis of HIV.