Potential for Virus Endogenization in Humans through Testicular Germ Cell Infection: the Case of HIV.

Viruses have colonized the germ line of our ancestors on several occasions during evolution, leading to the integration in the human genome of viral sequences from over 30 retroviral groups and a few nonretroviruses. Among the recently emerged viruses infecting humans, several target the testis (e.g., human immunodeficiency virus [HIV], Zika virus, and Ebola virus). Here, we aimed to investigate whether human testicular germ cells (TGCs) can support integration by HIV, a contemporary retrovirus that started to spread in the human population during the last century. We report that albeit alternative receptors enabled HIV-1 binding to TGCs, HIV virions failed to infect TGCs in vitro Nevertheless, exposure of TGCs to infected lymphocytes, naturally present in the testis from HIV+ men, led to HIV-1 entry, integration, and early protein expression. Similarly, cell-associated infection or bypassing viral entry led to HIV-1 integration in a spermatogonial cell line. Using DNAscope, HIV-1 and simian immunodeficiency virus (SIV) DNA were detected within a few TGCs in the testis from one infected patient, one rhesus macaque, and one African green monkey in vivo Molecular landscape analysis revealed that early TGCs were enriched in HIV early cofactors up to integration and had overall low antiviral defenses compared with testicular macrophages and Sertoli cells. In conclusion, our study reveals that TGCs can support the entry and integration of HIV upon cell-associated infection. This could represent a way for this contemporary virus to integrate into our germ line and become endogenous in the future, as happened during human evolution for a number of viruses.IMPORTANCE Viruses have colonized the host germ line on many occasions during evolution to eventually become endogenous. Here, we aimed at investigating whether human testicular germ cells (TGCs) can support such viral invasion by studying HIV interactions with TGCs in vitro Our results indicate that isolated primary TGCs express alternative HIV-1 receptors, allowing virion binding but not entry. However, HIV-1 entered and integrated into TGCs upon cell-associated infection and produced low levels of viral proteins. In vivo, HIV-1 and SIV DNA was detected in a few TGCs. Molecular landscape analysis showed that TGCs have overall weak antiviral defenses. Altogether, our results indicate that human TGCs can support HIV-1 early replication, including integration, suggesting potential for endogenization in future generations.

MicroRNAs in Sertoli cells: implications for spermatogenesis and fertility.

In recent decades, infertility has been considered a major widespread public health issue of very high concern. Currently, almost 50% of infertility cases are due to male factors, including semen disorders, obstructions, cryptorchidism, varicocele and testicular failures, which can occur due to malfunctions in both somatic and germ cells. In this context, besides other approaches, different miRNAs have been used as biomarkers for the diagnosis of male infertility, with different pathologic conditions such as Sertoli cell-only syndrome, mixed atrophy, and germ cell arrest. However, most studies related to male fertility do not point out the functions and cell targets of the described miRNAs. Initial investigations using experimental assays in murine and porcine models were performed, providing the first evidence of the influence of miRNAs on Sertoli cell function including, for instance, proliferation, maturation and hormone responses of these cells. The aim of this mini-review is therefore to summarize our present knowledge of this relevant subject and to highlight the importance of future investigations concerning the miRNA influence in the control of Sertoli cells, spermatogenesis and male fertility.

The spermatogonial stem cell niche in the collared peccary (Tayassu tajacu).

In the seminiferous epithelium, spermatogonial stem cells (SSCs) are located in a particular environment called the "niche" that is controlled by the basement membrane, key testis somatic cells, and factors originating from the vascular network. However, the role of Leydig cells (LCs) as a niche component is not yet clearly elucidated. Recent studies showed that peccaries (Tayassu tajacu) present a peculiar LC cytoarchitecture in which these cells are located around the seminiferous tubule lobes, making the peccary a unique model for investigating the SSC niche. This peculiarity allowed us to subdivide the seminiferous tubule cross-sections in three different testis parenchyma regions (tubule-tubule, tubule-interstitium, and tubule-LC contact). Our aims were to characterize the different spermatogonial cell types and to determine the location and/or distribution of the SSCs along the seminiferous tubules. Compared to differentiating spermatogonia, undifferentiated spermatogonia (A(und)) presented a noticeably higher nuclear volume (P < 0.05), allowing an accurate evaluation of their distribution. Immunostaining analysis demonstrated that approximately 93% of A(und) were GDNF receptor alpha 1 positive (GFRA1(+)), and these cells were preferentially located adjacent to the interstitial compartment without LCs (P < 0.05). The expression of colony-stimulating factor 1 was observed in LCs and peritubular myoid cells (PMCs), whereas its receptor was present in LCs and in GFRA1(+) A(und). Taken together, our findings strongly suggest that LCs, different from PMCs, might play a minor role in the SSC niche and physiology and that these steroidogenic cells are probably involved in the differentiation of A(und) toward type A(1) spermatogonia.

Sertoli cell proliferation in the adult testis is induced by unilateral gonadectomy in African catfish.

Survival and development of male germ cells depends on their close contact with Sertoli cells. In the cystic spermatogenesis found in fish, one germ cell clone, initially a single undifferentiated spermatogonium type A, is enclosed by and accompanied through spermatogenesis by a group of Sertoli cells. Previous work showed that after forming such spermatogenic cysts, Sertoli cells proliferated mainly during the mitotic expansion of the spermatogonial clone in the cyst. Here, we used unilateral gonadectomy (ULG) as experimental model to study Sertoli cell proliferation at the start of cyst development in adult African catfish testis. Four days after surgery, we observed a particularly strong increase in the number of mitotic Sertoli cells along with a significant increase in the number of mitotic single type A spermatogonia. Proliferation of pairs of spermatogonia or of larger germ cell clones, however, did not change. At the same time, pituitary transcript levels of the three gonadotropin-subunits (cga, glycoprotein hormones, alpha polypeptide; fshb, follicle stimulating hormone, beta polypeptide; lhb, luteinizing hormone, beta polypeptide) were not different between sham-operated and ULG males. However, expression of the gonadotropin-releasing hormone receptor gene gnrhr1 was significantly reduced after ULG, and Lh plasma levels were slightly elevated. In the testis remaining after ULG, Fsh receptor (fshr) mRNA levels increased significantly but luteinizing hormone/choriogonadotropin receptor (lhcgr) mRNA levels did not change. Circulating androgen levels did not differ between groups, but testicular androgen release increased significantly 2- to 3-fold after ULG. Considering the strong steroidogenic potency of Fsh and the expression of the fshr gene by Leydig cells in catfish, we explain the absence of an effect of ULG on circulating androgen levels by an Fshr-mediated, compensatory increase in the steroid production of the remaining testis, perhaps supported in addition by the increased Lh plasma levels. Since Fsh is a major stimulator of mammalian Sertoli cell proliferation, we propose that ULG-induced activation of the Fsh signalling system also promoted Sertoli cell proliferation and - possibly as a consequence of that - proliferation of single type A spermatogonia, providing the basis for an increased spermatogenic capacity.

Atrazine impairs testicular function in BalB/c mice by affecting Leydig cells.

Several studies have reported the effects of atrazine on the gonads of many experimental models. However, the short-term effects of in vivo exposure to atrazine on the testes of mice are not well clarified. Here we reported that adult BalB/c mice exposed to atrazine (50 mg kg-1 body weight) by gavage for three consecutive days have reduced numbers of 3ß-hydroxysteroid dehydrogenase positive Leydig cells (LCs), associated with increased in situ cell death fluorescence and caspase-3 immuno-expression in the testes. Consequently, immunostaining for cell cycle gene regulators showed increased expressions of p45, accompanied with increased expressions of cyclin D2 and E2. Histological observations of the gonads showed reduced number of germ cells in particular areas, sloughed seminiferous epithelium, presence of giant apoptotic cells close to the seminiferous tubule lumen and in the epididymal lumen along with low numbers of Leydig cells in the testicular interstitial areas. Similarly, LCs isolated from the testes of BalB/c mice that were exposed to atrazine (0.5, 25, 50 mg kg-1 body weight) in the same manner as in the first experiment presented dose-dependent increased caspase-3 activity, decreased cell viability, intratesticular and serum testosterone concentrations and LCs testosterone secretion. In summary, atrazine appears to directly decrease the number of testosterone secreting LCs in mice through apoptosis.

Effects of mild calorie restriction on reproduction, plasma parameters and hepatic gene expression in mice with altered GH/IGF-I axis.

The somatotropic axis, the hypothalamic-pituitary-gonadal axis and the nutritional status are deeply interrelated in mammals. Calorie restriction (CR) prolongs lifespan, but usually at some cost to reproduction. Interestingly, many of the physiological characteristics of animals with interruption in the somatotropic axis are shared by CR animals. The level of CR in most studies is 30-60%. We tested if a milder (20%) CR would promote health benefits without inhibiting reproduction in four types of mice with altered somatotropic axis: Ames dwarfs, GHR-KO, and PEPCK-bGH and MT-bGH transgenics. Fertility was not altered by CR in any of the examined groups. Mild CR did not affect final body weights or relative reproductive organ weights; did not alter plasma levels of glucose, insulin, IGF-I, testosterone, progesterone or estradiol; and did not influence hepatic expression of genes related to longevity. Altered activity of the GH/IGF-I axis in the different mice models studied had a major impact on the parameters analyzed. This preliminary study encourages speculation that mild regimens of CR can produce health and longevity benefits without the "costs" of impaired reproductive potential.

Prenatal plus postnatal exposure to Di(n-Butyl) phthalate and/or flutamide markedly reduces final sertoli cell number in the rat.

Androgens may be important regulators of Sertoli cell (SC) proliferation perinatally, with implications for the testicular dysgenesis syndrome (TDS) hypothesis. Fetal exposure of rats to 500 mg/kg . d di(n-butyl) phthalate (DBP) reduces fetal testosterone production and SC number at birth, but SC number recovers to normal by postnatal d (Pnd)25. It is unclear when and how SC proliferation is affected prenatally by DBP exposure or when and how postnatal compensation occurs. This study addressed these questions and investigated whether continued maternal exposure to DBP or to flutamide from Pnd1-Pnd15 could prevent SC number compensation, because this would have implications for how sperm counts might be lowered in TDS. DBP exposure attenuated SC proliferation by 7-18% throughout embryonic d (e)15.5-e21.5 (P < 0.05 at e21.5). After birth, SC proliferation increased significantly (>1.5-fold) between Pnd6 and Pnd10 in prenatally DBP-exposed animals, explaining the compensation. Continued maternal administration of DBP after birth attenuated (19% reduction) SC number compensation at Pnd25 and maternal administration of flutamide (100 mg/kg . d) to prenatally DBP-exposed animals was even more effective (42% reduction), suggesting the postnatal compensatory increase in SC proliferation after prenatal DBP exposure is androgen dependent. SC maturation (Pnd25) was unaffected, based on analysis of expression of key proteins, but lumen formation/expansion was attenuated in parallel with treatment-induced reduction in SC number. Our results provide further evidence that perinatal SC proliferation is androgen dependent and, importantly, show that similar exposure of mothers to antiandrogenic chemicals before birth and during lactation reduces final SC number, with implications for the origin of low sperm counts in TDS.