The blood testis barrier (BTB) was first identified by Dr Brian Setchel.
The BTB is made up of several different barriers. The peritubular myoid cells and the basement membrane of the seminiferous tubules forms one part and the tight junctions of the Sertoli cells form the other. Spermatogonia are outside this latter barrier; spermatocytes are on the other. Coordination of release and reattachment is required for progression of spermatogenesis from one stage to the next. This coordination is particularly important at stage VIII of the spermatogenic cycle. Failure at this stage results in arrest at the preleptotine spermatocyte stage. Actin based cytoskeletal components especially actin microfilaments are important in this and actin binding proteins are involved. (Li et al 2016).
Nan Li N, Tang EI, Yan Cheng C. Regulation of blood–testis barrier by actin binding proteins and protein kinases. Reproduction 2016; 151: R29–R41
Spermatogenesis is the production of spermatozoa within seminiferous tubules. The cells of spermatogenesis are
- Spermatogonia
- A (dense) spermatogonia
- A (pale) spermatogonia
- B spermatogonia
- Spermatocytes
- leptotene
- zygotene
- pachytene
- Spermatids
- round
- elongate
Spermatogenesis occurs in recognised stages. Most species have 12 (numbered I to XII) - dogs have 8.
Soares JM1, Avelar GF, França LR. The seminiferous epithelium cycle and its duration in different breeds of dog (Canis familiaris). J Anat 2009; 215: 462-471.
Spermatogenesis is a very intricate and closely regulated process. Because it is important to produce spermatozoa without cytogenetics or genetic abnormalities, there is tight regulation and apoptosis is a well-recognised phenomenon. Fas -FasL mediated apoptosis occurs to spermatogonia and all cells of the spermatogenic cycle and there are anti-apoptotic substances that create a balance between the two.
There is much intricate control with cross talk between all the components - spermatogonial stem cells, myoid cells, Sertoli cells, vascular endothelium, macrophages and interstitial endocrine cells.
Aitken RJ, Findlay JK, Hutt KJ, Kerr JB. Apoptosis in the germ line. Reproduction. 2011; 141: 139-150.
Giampietri C, Petrungaro S, Coluccia P, D'Alessio A, Starace D, Riccioli A, Padula F, Palombi F, Ziparo E, Filippini A, De Cesaris P. Germ cell apoptosis control during spermatogenesis. Contraception. 2005; 72: 298-302.
Potter SJ, DeFalco T. Role of the testis interstitial compartment in spermatogonial stem cell function. Reproduction. 2017; 153: R151-162.
The testis is a unique environment because it has a high metabolic demand yet has stable blood flow. The balance between free radical scavengers and reactive species is tenuous. A slight change results in oxidative stress.
Reactive oxygen species and then reactive nitrogen species play a role in the pathophysiology of the the testis. Hypoxic inducible factor (HIF) 1 is released in hypoxic conditions and this stimulates the formation of vascular endothelial growth factors like VEGF that induces angiogenesis and neovascularization. Release of HIF 1 occurs in systemic hypoxia and in localised hypoxia like varicocele and torsion.
Aitken RJ, Bromfield EG, Gibb Z. Oxidative stress and reproductive function: The impact of oxidative stress on reproduction: a focus on gametogenesis and fertilization. Reproduction. 2022; 164: F79-F94.
Aitken RJ. Impact of oxidative stress on male and female germ cells: implications for fertility. Reproduction. 2020; 159: R189-R201. doi: 10.1530/REP-19-0452. PMID:31846434.
Reyes JG, Farias JG, Henríquez-Olavarrieta S, Madrid E, Parraga M, Zepeda AB, Moreno RD. The hypoxic testicle: physiology and pathophysiology. Oxid Med Cell Longev. 2012; 929285
Turner TT, Lysiak JJ. Oxidative stress: a common factor in testicular dysfunction. J Androl. 2008: 29: 488-498.
Peritubular cells
Peritubular cells are barely visible in most testes. They are integral to paracrine signalling within the testis. When spermatogenesis is impared, there are changes in these cells and there are deposits of extracellular matrix.
These cells stain with smooth muscle actin and desmin, and this expression seems to be stimulated by testosterone. They act by moving the nonmotile spermatozoa along the seminiferous tubule.
There is considerable cross talk between these cells and interstitial endocrine cells and Sertoli cells. Without a signal from peritubular cells, interstitial endocrine cells disappear.
Peritubular cells produce decorin, a substance involved in deposition of extracellular matrix and with inflammation. Both the Sertoli cell and the peritubular cell is responsible for production of the basement membrane.
Mayerhofer A. (2013) Human testicular peritubular cells: more than meets the eye. Reproduction. 2013; 145: R107-R116.
Interstitial cells
Interstitial cells of the testis produce testosterone as a major steroid hormone, and cytokines for local regulation. The stimulus for testosterone production is the pulsatile release of luteinizing hormone from the pituitary. Production of testosterone in the testicular interstitial cell involves cholesterol formation, movement of cholesterol to the mitochondria, conversion of pregnenolone and production in of testosterone in the smooth endoplasmic reticulum. LH is important to this production.
Oxidative stress and its effects on interstitial cells results in altered LH mediated processes and causes reduced testosterone production. This occurs with aging.
Wang Y, Chen F, Ye L, Zirkin B, Chen H. Steroidogenesis in Leydig cells: effects of aging and environmental factors. Reproduction 2017; 154: R111-R122.
Testicular macrophages
As time goes on, the knowledge of the functions and activities of macrophages and dendritic cells of the testis improve and change. Most of the research is done in mice.
There are 2 main populations of macrophages. There are peritubular macrophages (MHCII+) near the myoid cells, and interstitial testicular macrophages (MHCII-) near the vessels and interstitial endocrine cells. There are different subpopulations of interstitial testicular macrophages. One type has activated genes to maintain immunosuppression.
Macrophages throughout the body are from 3 sources - early yolk sac macrophages, fetal liver monocytes, or bone marrow - derived monocytes. Testicular macrophages first appear at the yolk sac stage and later, have contributions from the fetal liver. There is no evidence yet that there is continuous replenishment from the bone marrow.
In inflammation, blood monocytes can contribute to inflammation.
Meinhardt A, Dejucq-Rainsford N, Bhushan S. Testicular macrophages: development and function in health and disease. Trends Immunol. 2021; 27: S1471-4906(21)00239-8.
The testis has a unique immunoregulatory environment and is said to have immune privilege or immune tolerance. This is a multifaceted process involving the physical barrier of the blood brain barrier, other innate factors and inhibition of adaptive immune function. This is not all passive (like the BTB), but there is active inhibition involving multiple mechanisms, including active local immunosuppression.
Adaptive immune inhibition involves Treg cells and inhibition of autoreactive T cells. Programmed death receptor (PD-1) and the PD ligand (PD-L1) are involved (Wang et al 2017). Spermatocytes and spermatids in mice constituitively express PD-L1, and Sertoli cells can be induced to express PD-L1.
There are Toll like receptors in the testis and inflammatory cytokines and inflammatory pathways exist. These are probably activated in systemic inflammation and result in alteration to testicular function by altering Sertoli cell function.
The normal testis is therefore generally anti-inflammatory and this is maintained by testosterone from the interstitial endocrine cells. When inflammation does occur, there is up-regulation of cytokines such as interleukin-1 beta, interleukin-1 alpha, interleukin-6 and tumour necrosis factor alpha. The attraction of lymphocytes in inflammation is mediated by CCL2, CCL3 and CCL4. These result in a pro-inflammatory cytokine environment with interferon alpha, interleukin-6 and TNF alpha being involved. Receptors for the cytokines are found on germ cells and with attachment of these cytokines, the germ cells undergo apoptosis.
The most common immune cell in the testis is the macrophage. Bhushan and Meinhardt (2017) wrote a review about them. Testicular macrophages are dominated by the M2 type, producing antiinflammatory cytokines like IL-10 and TGF Beta.
Chen et al (2022) reviewed immune homeostatis and disorders in the Testis.
Bhushan S, Meinhardt A. The macrophages in testis function. J Reprod Immunol. 2017; 119:107-112
Chen R, Wang F, Chen Y, Han D. Immune homeostasis and disorder in the testis - Roles of Sertoli cells. J Reprod Immunol. 2022; 152: 103625.
Guazzone VA, Jacobo P, Theas MS, Lustig L. Cytokines and chemokines in testicular inflammation: A brief review. Microsc Res Tech. 2009; 72: 620-628
Hedger MP. (2011) Toll-like receptors and signalling in spermatogenesis and testicular responses to inflammation--a perspective. J Reprod Immunol. 2011; 88: 130-141.
Wang LL, Li ZH, Hu XH, Muyayalo KP, Zhang YH, Liao AH. The roles of the PD-1/PD-L1 pathway at immunologically privileged sites. Am J Reprod Immunol. 2017; 78:
Immune privilege
Most of the germ cells are formed after the immune system matures and the cells that develop are not included in self tolerance.
The blood testis barrier is important in preventing autoimmune reactions against cells of spermatogenesis. The blood testis barrier is formed by junctional complexes in Sertoli cells and there are junctional adhesion molecules including claudins and occludin.
The interstitium of the testis is hyporesponsive to immune stimuli and is immunotolerant. Testosterone is important in this.
Peritubular cells and especially myoid cells are important in providing antiinflammatory cytokines such as TGF B, macrophage chemotactic protein 1, and leukemia inhibiting factor.
Interstitial endocrine cells produce testosterone in high concentrations and this high concentration is important in the hyporesponsiveness of the testis by causing a reduction in the production of inflammatory cytokines like IL-1, IL-6 and TNF alpha.
Testosterone has a direct effect on peritubular and myoid cells and their expression of TNF-alpha. There was no effect on testicular macrophages. Testosterone influence CD4+CD25+Foxp3+ regulatory T cells (Treg cells) and thereby modulate inflammation. This is by direct action in stimulating secretion of IL-10 and TGF beta via androgen receptors on CD4+ T cells. It also inhibits the secretion of proinflammatory IL-1beta, IL-6 and TNF-alpha. Testosterone is thus antiinflammatory and inhibits anti autoimmune reactions.
Testicular interstitial macrophages are in large numbers and are in close association with interstitial endocrine cells. They are continuously repopulated from blood monocytes. They also influence Sertoli cell function by soluble mediators. They have immunosupressive characteristics. Some macrophages have proinflammatory functions and these populate the testis when stimulation occurs, such as LPS administration. Cytokines probably regulate macrophage function.TGF beta and activin A probably inhibit immune responses.
Spermatogenic cells dont display MHCII, this potentially avoid direct recognition by CD4 and CD8 cells. Ducts dont express MHCII in the normal situation.
Costimulatory molecules appear to be absent in the testis.
Dendritic cells appear to maintain immune privilge until there is testicular injury.
Mast cells and their distribution are important in the inflammatory reaction. They are found around blood vessels and close to the tunica albuginea. Fibrosis of the testis is probably a mast cell mediated condition, but their restricted distribution is probably protective.
There are therefore multiple levels of regulation of immune function - there is built in redundancy.
Winnall WR, Hedger MP. (2013) Phenotypic and functional heterogeneity of the testicular macrophage population: a new regulatory model. J Reprod Immunol 2013; 97: 147-158.
Fijak M , Damm L-J, Wenzel1 J-P, Aslani F, Walecki M, Wahle E , Eisel F, Bhushan S, Hackstein H , Baal N , Schuler G, Konrad L, Rafiq A, O’Hara L, Smith LB, Meinhardt A. Influence of Testosterone on Inflammatory Response in Testicular Cells and Expression of Transcription Factor Foxp3 in T Cells. Amer J Reprod Immunol 2015; 74: 12-25.
Antisperm antibody
It is well known that spermatozoa are antigenic and stimulate an adaptive immune response particularly with the formation of antisperm antibody. The appearance of ASA in serum is present in males and in females but it is not always associated with infertility, in fact many individuals with ASA in serum are fertile. It is the presence of antisperm antibody within the reproductive tract and particularly when this antibody inhibits motility or stimulates complement activation.
A. Hasegawa, H. Shibahara. (2014) The history and current status of anti-sperm antibody research. J Reprod Immunol 2014; 106:
Fijak M, Meinhardt A (2006) The testis in immune priviledge. Immunological reviews 213: 66-81
Epididymis
The function of the epididymis is to take testicular spermatozoa, which are unable to move or fertilise an oocyte and to produce a fully functional spermatozoon. This involves "differentiation" so that the spermatozoa and can move, bind to the oocyte membrane and to fuse with the oocyte. This requires sequential modification through the epididymis. The epididymis also concentrates the spermatozoa as rete testis fluid has a high volume and low concentration.
Dacheux JL, Dacheux F. (2014) New insights into epididymal function in relation to sperm maturation. Reproduction. 2013; 19; 147: R27-R42.