Primate Testis Research Articles

Testis-specific H2B.W1 disrupts nucleosome integrity by reducing DNA-histone interactions.

Multiple testis-specific histone variants are involved in the dynamic chromatin transitions during spermatogenesis. H2B.W1 (previously called H2BFWT) is an H2B variant specific to primate testis with hitherto unclear functions, although its single-nucleotide polymorphisms (SNPs) are closely associated with male non-obstructive infertility. Here, we found that H2B.W1 is only expressed in the mid-late spermatogonia stages, and H2B.W1 nucleosomes are defined by a more flexible structure originating from weakened interactions between histones and DNA. Furthermore, one of its SNPs, H2B.W1-H100R, which is associated with infertility, further destabilizes the nucleosomes and increases the nucleosome unwrapping rate by interfering with the R100 and H4 K91/R92 interaction. Our results suggest that destabilizing H2B.W1 containing nucleosomes might change the chromatin structure of spermatogonia, and that H2B.W1-H100R enhances the nucleosome-destabilizing effects, leading to infertility.

Testis-on-chip platform to study ex vivo primate spermatogenesis and endocrine dynamics

Here, we report a testis-on-chip platform for the ex vivo culture of seminiferous tubules isolated from human and non-human primate testis. Tissues are cultured in a dedicated chamber with continuous perfusion via a vascular-like channel. The platform is fabricated from PDMS using a 3D printed mold, after design has been optimized, e.g., for the barrier between the culture chamber and the perfusion channel. COMSOL modeling revealed no direct negative impact of the flow on the tissues for the applied flowrate in the device, shear rate remaining in the physiological range. Culture experiments were performed using adult human seminiferous tubules from gender dysphoria patients and prepubertal seminiferous tubules from a 6-month-old marmoset for up to 11 and 9 days, respectively. First, microscopic, and live imaging revealed the presence of viable cell populations in both types of samples. Next, marmoset tissues were exposed to stimulatory conditions through perfusion of gonadotropins at different doses. The tissue response was characterized by histological analysis after their recovery from the device and testosterone and estradiol analysis in the effluent. Histological observations suggested improved maintenance of marmoset testicular tissues under stimulatory conditions, which similarly resulted in an increase in both testosterone and estradiol production, with yet different patterns for the low-dose and high-dose stimulation. The herein reported testis-on-chip platform shows great promise to evaluate endocrine and toxic effects on the testis.

The in-vitro effect of gonadotropin-releasing hormones, GnRH-I and GnRH-II, on the motility, vitality and acrosome integrity of Vervet monkey (Chlorocebus aethiops) spermatozoa.

Two isoforms of the gonadotropin-releasing hormone (GnRH), GnRH-I and GnRH-II, are expressed in mammals, and the presence of one or more GnRH-like peptides has been demonstrated in the male reproductive tract. GnRH and its receptors (GnRHR) are present in human and non-human primate testis, prostate, epididymis, seminal vesicle, spermatozoa and seminal human plasma. GnRH-II is site-specific and acts directly in an inhibitory or stimulatory fashion. Previous studies speculated that GnRH-II could disrupt specific sperm processes, such as sperm motility or capacitation and could be utilized as an effective contraceptive agent. Our study aimed to investigate the in-vitro effects of GnRH-I and GnRH-II on Vervet monkey sperm function. Electro-ejaculated semen samples from 10 Vervet monkeys (Chlorocebus aethiops) were used to select motile sperm populations. Sperm aliquots were incubated with GnRH-I and GnRH-II at different concentrations for 1 h, where after sperm motility and kinematic parameters were assessed using the automated Sperm Class Analyser. Additional sperm aliquots were incubated with two 10-amino acid control peptides, a non-related peptide and an inactive peptide to exclude the possible influence on sperm motility from other peptides with a structure similar to GnRH. Additionally, a GnRHR-I antagonist (GnRHR-A), Cetrorelix, was tested to establish its antagonistic capability on GnRH. The effect of selected concentrations of GnRH-I and GnRH-II on sperm vitality and acrosome intactness was also evaluated after 10- and 60min exposure. Analysis of the percentage total sperm motility revealed that different concentrations for GnRH-I and GnRH-II inhibited sperm motility significantly. While sperm progressiveness was also notably affected and a trend of decreased sperm kinematics were evident, no effect was found on sperm vitality or acrosome intactness. The non-related and inactive peptides had no impact on sperm motility. The GnRHR-A demonstrated no effect on sperm motility and effectively blocked the inhibitory outcome on the motility of both GnRH isoforms. While GnRH-I or GnRH-II at low-dose concentrations resulted in in-vitro inhibition of sperm motility, it appears to have no adverse effects on other sperm functional parameters evaluated. These collective observations possibly indicate an essential role for GnRH in the in-vivo process of sperm selection in the female reproductive tract.

Limited spermatogenic differentiation of testicular tissue from prepubertal marmosets (Callithrix jacchus) in an in vitro organ culture system

Purposeof the research: To achieve male fertility preservation and restoration, experimental strategies for in vitro germ cell differentiation are required. The effects of two different culture conditions on in vitro maintenance and differentiation of non-human primate germ cells was studied. Three testes from three 6-month-old marmosets were cultured using a gas-liquid interphase system for 12 days. Testicular maturation in pre-culture control and samples cultured in gonadotropin and serum supplemented and non-supplemented culture samples was evaluated using Periodic Acid-Schiff (PAS) and immunohistochemical stainings. Principle resultsGonadotropins and serum-supplemented tissues demonstrate up to meiotic differentiation (BOULE + Pachytene spermatocyte) and advanced localization of germ cells (MAGEA4+). Moreover, complex (with gonadotropin and marmoset monkey serum) conditions induced progression in somatic cell maturation with advanced seminiferous epithelial organization, maintenance of encapsulation of cultured fragments with peritubular-myoid cells, preservation of tubular structural integrity and architecture. Major conclusionsWe report stimulation-dependent in vitro meiotic transition in non-human primate testes. This model represents a novel ex vivo approach to obtain crucial developmental progression.

The expression of type I and II gonadotropin-releasing hormone receptors transcripts in Vervet monkey (Chlorocebus aethiops) spermatozoa

Gonadotropin-Releasing Hormone (GnRH), acting via the GnRH receptor (GnRHR), and a member of G-protein coupled receptor (GPCR), plays an essential role in the control of reproduction while operating primarily at the hypothalamic level of the gonadotropic axis. GnRH and its receptor are co-expressed in certain specific cells, suggesting an autocrine regulation of such cells. In the male reproductive system, two forms of GnRH (I and II) and its receptors (GnRHR) are present in the human and non-human primate (NHP) testis, prostate, epididymis, seminal vesicle, and human spermatozoa. In humans, the GnRHR-II receptor gene is disrupted by a frameshift in exon 1 and a stop codon in exon 2, rendering the receptor non-functional, whereas a fully functional GnRHR-II receptor is present in New-World and Old-World monkeys. There is no evidence of the existence of a GnRH receptor in NHP sperm. Since the NHP has a phylogenetic relationship to man and is often used as models in reproductive physiology, this present study aimed to determine GnRHR-I and GnRHR-II in Vervet monkey (Chlorocebus aethiops) spermatozoa. A total of 24 semen samples were obtained from four adult Vervet monkeys through electro-ejaculation and utilized for genotyping and gene expression analysis of GnRHR-I and II. Here we report that both receptors were successfully identified in the Vervet monkey sperm with the abundance of GnRHR-I gene expression compared to GnRHR-II. In comparison to the human, there is no evidence of such a stop codon at position 179 in exon 2 of the Vervet GnRHR-II. These findings suggest that both receptors are transcriptionally functional in Vervet spermatozoa.

SIRT1 Expression and Regulation in the Primate Testis.

The epigenetic mechanisms controlling germ cell development and differentiation are still not well understood. Sirtuin-1 (SIRT1) is a nicotinamide adenosine dinucleotide (NAD)-dependent histone deacetylase and belongs to the sirtuin family of deacetylases. It catalyzes the removal of acetyl groups from a number of protein substrates. Some studies reported a role of SIRT1 in the central and peripheral regulation of reproduction in various non-primate species. However, testicular SIRT1 expression and its possible role in the testis have not been analyzed in primates. Here, we document expression of SIRT1 in testes of different primates and some non-primate species. SIRT1 is expressed mainly in the cells of seminiferous tubules, particularly in germ cells. The majority of SIRT1-positive germ cells were in the meiotic and postmeiotic phase of differentiation. However, SIRT1 expression was also observed in selected premeiotic germ cells, i.e., spermatogonia. SIRT1 co-localized in spermatogonia with irisin, an endocrine factor specifically expressed in primate spermatogonia. In marmoset testicular explant cultures, SIRT1 transcript levels are upregulated by the addition of irisin as compared to untreated controls explants. Rhesus macaques are seasonal breeders with high testicular activity in winter and low testicular activity in summer. Of note, SIRT1 mRNA and SIRT1 protein expression are changed between nonbreeding (low spermatogenesis) and breeding (high spermatogenesis) season. Our data suggest that SIRT1 is a relevant factor for the regulation of spermatogenesis in primates. Further mechanistic studies are required to better understand the role of SIRT1 during spermatogenesis.

The Glucocorticoid Receptor NR3C1 in Testicular Peritubular Cells is Developmentally Regulated and Linked to the Smooth Muscle-Like Cellular Phenotype.

Whether glucocorticoids (GC) can directly affect human testicular functions is not well understood. A predominant site of GC receptor (GR; NR3C1) expression in the adult testis are peritubular smooth muscle-like cells, which express smooth muscle actin (ACTA2), contract and thereby are involved in sperm transport. In contrast to the adult, neither GR nor ACTA2, or elastin (ELN) were detected in the peritubular compartment before puberty in non-human primate testes. In isolated human testicular peritubular cells (HTPCs), activation of GR by dexamethasone (Dex) caused the translocation of GR to the nucleus and stimulated expression of ACTA2 and ELN, without affecting the expression of collagens. Cytoskeletal ACTA2-rearrangements were observed and were associated with an increased ability to contract. Our results indicate post-pubertal testicular roles of GC in the maintenance of the contractile, smooth muscle-like phenotype of peritubular cells.

Estrogens in Human Male Gonadotropin Secretion and Testicular Physiology From Infancy to Late Puberty.

Several reports in humans as well as transgenic mouse models have shown that estrogens play an important role in male reproduction and fertility. Estrogen receptor alpha (ERα) and beta (ERβ) are expressed in different male tissues including the brain. The estradiol-binding protein GPER1 also mediates estrogen action in target tissues. In human testes a minimal ERα expression during prepuberty along with a marked pubertal up-regulation in germ cells has been reported. ERβ expression was detected mostly in spermatogonia, primary spermatocytes, and immature spermatids. In Sertoli cells ERβ expression increases with age. The aromatase enzyme (cP450arom), which converts androgens to estrogens, is widely expressed in human tissues (including gonads and hypothalamus), even during fetal life, suggesting that estrogens are also involved in human fetal physiology. Moreover, cP450arom is expressed in the early postnatal testicular Leydig cells and spermatogonia. Even though the aromatase complex is required for estrogen synthesis, its biological relevance is also related to the regulation of the balance between androgens and estrogens in different tissues. Knockout mouse models of aromatase (ArKO) and estrogen receptors (ERKOα, ERKOβ, and ERKOαβ) provide an important tool to study the effects of estrogens on the male reproductive physiology including the gonadal axis. High basal serum FSH levels were reported in adult aromatase-deficient men, suggesting that estrogens are involved in the negative regulatory gonadotropin feedback. However, normal serum gonadotropin levels were observed in an aromatase-deficient boy, suggesting a maturational pattern role of estrogen in the regulation of gonadotropin secretion. Nevertheless, the role of estrogens in primate testis development and function is controversial and poorly understood. This review addresses the role of estrogens in gonadotropin secretion and testicular physiology in male humans especially during childhood and puberty.

Microfluidics in male reproduction: is ex vivo culture of primate testis tissue a future strategy for ART or toxicology research?

The significant rise in male infertility disorders over the years has led to extensive research efforts to recapitulate the process of male gametogenesis in vitro and to identify essential mechanisms involved in spermatogenesis, notably for clinical applications. A promising technology to bridge this research gap is organ-on-chip (OoC) technology, which has gradually transformed the research landscape in ART and offers new opportunities to develop advanced in vitro culture systems. With exquisite control on a cell or tissue microenvironment, customized organ-specific structures can be fabricated in in vitro OoC platforms, which can also simulate the effect of in vivo vascularization. Dynamic cultures using microfluidic devices enable us to create stimulatory effect and non-stimulatory culture conditions. Noteworthy is that recent studies demonstrated the potential of continuous perfusion in OoC systems using ex vivo mouse testis tissues. Here we review the existing literature and potential applications of such OoC systems for male reproduction in combination with novel bio-engineering and analytical tools. We first introduce OoC technology and highlight the opportunities offered in reproductive biology in general. In the subsequent section, we discuss the complex structural and functional organization of the testis and the role of the vasculature-associated testicular niche and fluid dynamics in modulating testis function. Next, we review significant technological breakthroughs in achieving in vitro spermatogenesis in various species and discuss the evidence from microfluidics-based testes culture studies in mouse. Lastly, we discuss a roadmap for the potential applications of the proposed testis-on-chip culture system in the field of primate male infertility, ART and reproductive toxicology.

Irisin is expressed by undifferentiated spermatogonia and modulates gene expression in organotypic primate testis cultures

The molecular mechanisms regulating undifferentiated spermatogonial cell proliferation and differentiation are still not fully understood. Irisin is an exercise-induced hormone, which is a cleaved and secreted fragment of the fibronectin type III repeat containing 5 (FNDC5) transmembrane protein. Recent studies have demonstrated the role of irisin in cell proliferation and differentiation in various tissues. However, testicular irisin expression and its potential action have not been analyzed. Here, we demonstrate expression of irisin in undifferentiated spermatogonia of primates and in the tree shrew, a bridging species between primates and insectivores. Rhesus monkeys are seasonal breeders with annual phases of high and low testicular activity and germ cell proliferation. Interestingly, expression of both FNDC5 mRNA and irisin is altered between breeding (high spermatogenesis) and nonbreeding seasons (low spermatogenesis). Organotypic testis culture in the presence of irisin increased the expression levels of the Sertoli cell (GDNF) and spermatogonial transcripts Kruppel-like factor 4 (KLF4), Inhibitor of differentiation 4 (ID4), Cluster of differentiation 117 (cKIT), and SALL4, compared to untreated controls, while irisin suppressed its own FNDC5 mRNA. Our data suggest that irisin is a novel endocrine factor involved in the regulation of spermatogonial activities in the testes of primates.

Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate.

STUDY QUESTIONCan full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously?SUMMARY ANSWERIntratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset (Callithrix jacchus) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months.WHAT IS KNOWN ALREADYAuto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful.STUDY DESIGN, SIZE, DURATIONIn this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8–1.0 mm3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice (n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4–6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation.PARTICIPANTS/MATERIALS, SETTING, METHODSTransplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids.MAIN RESULTS AND THE ROLE OF CHANCEFour months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered (P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was established. Overall, 78% of the intratesticular transplants showed post-meiotic differentiation (round spermatids, elongating/elongated spermatids and spermatozoa). However, during the same period, spermatocytes (early meiotic germ cells) were the most advanced germ cell type present in the ectopic transplants. Nine months post-transplantation, 50% of the intratesticular transplants survived, whilst none of the ectopic transplants was recovered (P < 0.0001). Transplants contained more spermatogonia per tubule (P = 0.018) than at 4 months. Complete spermatogenesis was observed in all recovered transplants (100%), indicating a progressive spermatogenic development in intratesticular transplants between the two time-points. Nine months post-transplantation, transplants contained more seminiferous tubules with post-meiotic germ cells (37 vs. 5%; P < 0.001) and fewer tubules without germ cells (2 vs. 8%; P = 0.014) compared to 4 months post-transplantation.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONAlthough xenotransplantation of marmoset ITT was successful, it does not fully reflect all aspects of a future clinical setting. Furthermore, due to ethical restrictions, we were not able to prove the functionality of the spermatozoa produced in the marmoset transplants.WIDER IMPLICATIONS OF THE FINDINGSIn this pre-clinical study, we demonstrated that testicular parenchyma provides the required microenvironment for germ cell differentiation and long-term survival of immature marmoset testis tissue, likely due to the favourable temperature regulation, growth factors and hormonal support. These results encourage the design of new experiments on human ITT xenotransplantation and show that intratesticular transplantation is likely to be superior to ectopic transplantation for fertility restoration following gonadotoxic treatment in childhood.STUDY FUNDING/COMPETING INTEREST(S)This project was funded by the ITN Marie Curie Programme ‘Growsperm’ (EU-FP7-PEOPLE-2013-ITN 603568) and the scientific Fund Willy Gepts from the UZ Brussel (ADSI677). D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

NLRP3 in somatic non-immune cells of rodent and primate testes.

NLRP3 is part of the NLRP3 inflammasome and a global sensor of cellular damage. It was recently discovered in rodent Sertoli cells. We investigated NLRP3 in mouse, human and non-human primate (marmoset and rhesus macaque) testes, employing immunohistochemistry. Sertoli cells of all species expressed NLRP3, and the expression preceded puberty. In addition, peritubular cells of the adult human testes expressed NLRP3. NLRP3 and associated genes (PYCARD, CASP1, IL1B) were also found in isolated human testicular peritubular cells and the mouse Sertoli cell line TM4. Male infertility due to impairments of spermatogenesis may be related to sterile inflammatory events. We observed that the expression of NLRP3 was altered in the testes of patients suffering from mixed atrophy syndrome, in which tubules with impairments of spermatogenesis showed prominent NLRP3 staining. In order to explore a possible role of NLRP3 in male infertility, associated with sterile testicular inflammation, we studied a mouse model of male infertility. These human aromatase-expressing transgenic mice (AROM+) develop testicular inflammation and impaired spermatogenesis during aging, and the present data show that this is associated with strikingly elevated Nlrp3 expression in the testes compared to WT controls. Interference by aromatase inhibitor treatment significantly reduced increased Nlrp3 levels. Thus, throughout species NLRP3 is expressed by somatic cells of the testis, which are involved in testicular immune surveillance. We conclude that NLRP3 may be a novel player in testicular immune regulation.

The C-X-C signalling system in the rodent vs primate testis: impact on germ cell niche interaction.

In zebrafish, action of the chemokine Cxcl12 is mediated through its G-protein-coupled seven-transmembrane domain receptor Cxcr4 and the atypical receptor Cxcr7. Employing this animal model, it was revealed that this Cxcl12 signalling system plays a crucial role for directed migration of primordial germ cells (PGC) during early testicular development. Importantly, subsequent studies indicated that this regulatory mechanism is evolutionarily conserved also in mice. What is more, the functional role of the CXCL12 system does not seem to be limited to early phases of testicular development. Data from mouse studies rather demonstrate that CXCL12 and its receptors are also involved in the homing process of gonocytes into their niches at the basal membrane of the seminiferous tubules. Intriguingly, even the spermatogonial stem cells (SSCs) present in the adult mouse testis appear to maintain the ability to migrate towards a CXCL12 gradient as demonstrated by functional in vitro migration assays and in vivo germ cell transplantation assays. These findings not only indicate a role of the CXCL12 system throughout male germ cell development in mice but also suggest that this system may be evolutionarily conserved. In this review, we take into account the available literature focusing on the localization patterns of the CXCL12 system not only in rodents but also in primates, including the human. Based on these data, we discuss whether the CXCL12 system is also conserved between rodents and primates and discuss the known and potential functional consequences.

Exposure to phthalate esters induces an autophagic response in male germ cells.

Phthalate esters are plasticizers that impart flexibility to polvinylchloride plastics. As they are not covalently bound, they can leach from a wide range of products, including food containers, medical devices, clothing, and toys, leading to widespread environmental exposure. Phthalate toxicity has been linked to male infertility by disrupting testosterone production and testis development. Phthalates also impair proliferation and viability of spermatogonial stem cells (SSC), the role of which is to support lifelong spermatogenesis. To elucidate cellular mechanisms in spermatogonia affected by long-term phthalate exposure, we grafted primate testis tissue into mice. Grafts treated with di-n-butyl phthalate showed an increase in autophagy compared to controls. Short term in vitro exposure of porcine germ cells to mono(2-ethylhexyl) phthalate, also resulted in an increase in autophagy. Viability was lower in cells exposed to phthalates, but treatment with rapamycin to induce autophagy significantly increased viability. The data suggests autophagy is triggered in spermatogonia as a response to a toxic insult, which may constitute a survival mechanism in spermatogonia.

Immunocytochemical localization of kisspeptin and kisspeptin receptor in the primate testis.

Hypothalamic kisspeptin-kisspeptin receptor signalling in primates ensures the successful progression into puberty during development and maintenance of reproductive capacity during adulthood. Human testis has been shown to express high-to-moderate levels of kisspeptin and kisspeptin receptor gene expression. In this study, we aimed at characterizing the localization of kisspeptin and kisspeptin receptor in adult primate testis tissue. Immunocytochemistry was performed on paraffin-embedded testicular sections from adult rhesus monkeys and from common marmoset monkeys. Kisspeptin receptor was detected in Sertoli cells in the periphery of the seminiferous tubules in adult testes of both species. In contrast, kisspeptin was not localized in the seminiferous epithelium and was detected only in the interstitial compartment of the adult rhesus monkey testis. Kisspeptin receptor and kisspeptin are localized in the testis of Old World and New World primates.

Developmental expression patterns of chemokines CXCL11, CXCL12 and their receptor CXCR7 in testes of common marmoset and human.

The chemokine receptor CXCR7 interacts with the chemokines CXCL11 and CXCL12. During development, this ligand receptor system (C-X-C) provokes cell-type-specific responses in terms of migration, adhesion or ligand sequestration. It is active in zebrafish and rodents but no data are available for its presence or function in primate testes. Real-time quantitative polymerase chain reaction was performed in monkeys to detect CXCL11, CXCL12 and CXCR7. At the protein level, CXCL12 and CXCR7 were localized in the testes of the marmoset (Callitrix jacchus) whereas CXCR7 patterns were determined for various stages in human testes. Morphometry and flow cytometry were applied to quantify CXCR7-positive cells in monkeys. Transcript levels and protein expression of CXCR7 were detectable throughout testicular development. In both species, CXCR7 protein expression was restricted to premeiotic germ cells. In immature marmoset testes, 69.9% ± 9% of the total germ cell population were labelled for CXCR7, whereas in the adult, 4.7% ± 2.7% were positive for CXCR7. CXCL12 mRNA was detectable in all developmental stages in marmosets. The CXCL12 protein was exclusively localized to Sertoli cells. This pattern of CXCL12/CXCR7 indicates their involvement in regulatory processes that possibly orchestrate the interaction between undifferentiated germ cells and Sertoli cells.

Simian immunodeficiency virus infection and immune responses in the pig-tailed macaque testis.

The testis is a site of immune privilege in rodents, and there is evidence that T cell responses are also suppressed in the primate testis. Local immunosuppression is a potential mechanism for HIV persistence in tissue reservoirs that few studies have examined. The response of the pig-tailed macaque testis to SIVmac239 infection was characterized to test this possibility. Testes were surgically removed during early-chronic (10 wk) and late-chronic (24-30 wk) SIV infection in 4 animals and compared with those from 7 uninfected animals. SIV infection caused only minor disruption to the seminiferous epithelium without marked evidence of inflammation or consistent changes in total intratesticular leukocyte numbers. Infection also led to an increase in the relative proportion of testicular effector memory CD8(+) T cell numbers and a corresponding reduction in central memory CD4(+) T cells. A decrease in the relative proportion of resident-type CD163(+) macrophages and DCs was also observed. SIV-specific CD8(+) T cells were detectable in the testis, 10-11 wk after infection by staining with SIV Gag-specific or Tat-specific MHC-I tetramers. However, testicular CD8(+) T cells from the infected animals had suppressed cytokine responses to mitogen activation. These results support the possibility that local immunosuppression in the testis may be restricting the ability of T cells to respond to SIV or HIV infection. Local immunosuppression in the testis may be an underexplored mechanism allowing HIV persistence.

Expression of the oestrogen receptor GPER by testicular peritubular cells is linked to sexual maturation and male fertility.

Besides the two nuclear oestrogen receptors (ESR1/ESR2), the G protein-coupled oestrogen receptor (GPER) was described in the human testis but little is known about testicular GPER during development or male infertility. We performed an immunohistochemical analysis using human and rhesus monkey testicular samples. The results obtained in adult primate testes showed GPER in interstitial and vascular cells as well as in smooth muscle-like peritubular cells, which build the wall of seminiferous tubules. Expression of GPER was also found in cultured human testicular peritubular cells (HPTCs) by Western blotting and RT-PCR/sequencing. Furthermore, as seen in time-lapse videos of cultured cells, addition of a specific GPER agonist (G1) significantly reduced the numbers of HTPCs within 24 h. A GPER antagonist (G15) prevented this action, implying a role for GPER related to the control of cell proliferation or cell death of peritubular cells. Peritubular cell functions and their phenotype change, for example, during post-natal development and in the cases of male infertility. The study of non-human primate samples revealed that GPER in peritubular cells was detectable only from the time of puberty onwards, while in samples from infantile and prepubertal monkeys only interstitial cells showed immunopositive staining. In testicular biopsies of men with mixed atrophy, a reduction or loss of immunoreactive GPER was found in peritubular cells surrounding those tubules, in which spermatogenesis was impaired. In other cases of impaired spermatogenesis, namely when the tubular wall was fibrotically remodelled, a complete loss of GPER was seen. Thus, the observed inverse relation between the state of fertility and GPER expression by peritubular cells implies that the regulation of primate testicular peritubular cells by oestrogens is mediated by GPER in both, health and disease.

Characterisation of macaque testicular leucocyte populations and T-lymphocyte immunity

The rodent testis is well established as a site of immune privilege where both innate and acquired immune responses are suppressed. Immune cells and responses within human or non-human primate testes, by contrast, are poorly characterised. This study used multi-colour flow cytometry to characterise the leukocytes in testicular cells isolated from 12 young adult pigtail macaques (Macaca nemestrina) by collagenase dispersal, and to measure the cytokine responses of macaque testicular T-lymphocytes to mitogens. B-lymphocytes and granulocytes were present in very low numbers (0.24% and 3.3% of leukocytes respectively), indicating minimal blood contamination. A median of 30.8% of the recovered testicular leukocytes were CD3+ lymphocytes, with CD4 and CD8 T-lymphocyte proportions similar to those in the blood. The proportion of naïve T-lymphocytes in the testis was low, with significantly higher frequencies of central memory cells, compared with the blood. A median of 42.7% of the testicular leukocytes were CD163+ macrophages, while 4.5% were CD14+CD163− monocyte-like macrophages. Small populations of myeloid and plasmacytoid dendritic cells, NK cells and NKT cells were also detected. Following mitogen stimulation, 19.7% of blood T-lymphocytes produced IFNγ and/or TNF, whereas significantly fewer (4.4%) of the testicular T-lymphocytes responded to stimulation. Our results characterise the immune cells within the adult macaque testis and identify a suppression of T-lymphocyte responses. This study provides a baseline to examine the immunology of the primate testis and suggests that testicular immune privilege could also be present in primates.

Kiss1 and Kiss1 receptor expression in the rhesus monkey testis: a possible local regulator of testicular function

Abstract