Neonatal Porcine Germ Cells Dedifferentiate and Display Osteogenic and Pluripotency Properties

Gonocytes are progenitors of spermatogonial stem cells in the neonatal testis. We have previously shown that upon culturing, neonatal porcine gonocytes and their colonies express germ cell and pluripotency markers. The objectives of present study were to investigate in vitro trans-differentiation potential of porcine gonocytes and their colonies into cells from three germinal layers, and to assess pluripotency of cultured gonocytes/colonies in vivo. For osteogenic and tri-lineage differentiation, cells were incubated in regular culture media for 14 and 28 days, respectively. Cells were cultured for an additional 14 days for osteogenic differentiation or 7 days for differentiation into derivates of the three germinal layers. Osteogenic differentiation of cells and colonies was verified by Alizarin Red S staining and tri-lineage differentiation was confirmed using immunofluorescence and gene expression analyses. Furthermore, upon implantation into recipient mice, the cultured cells/colonies developed teratomas expressing markers of all three germinal layers. Successful osteogenic differentiation from porcine germ cells has important implications for bone regeneration and matrix formation studies. Hence, gonocytes emerge as a promising source of adult pluripotent stem cells due to the ability to differentiate into all germinal layers without typical biosafety risks associated with viral vectors or ethical implications.

Vapor cannabis exposure generationally affects male reproductive functions in mice

This study was performed to examine whether vapor exposure to cannabis plant matter negatively impacts male reproductive functions and testis development in mice. Adult CD-1 male mice (F0) were exposed to air (control) or 200 mg of vaporized cannabis plant matter 3x/day over a 10 day period. Subsequently, F0 males were bred with drug naive CD-1 females to generate F1 males, and F1 offspring were used to generate F2 males. Cannabis vapor exposure decreased sperm count and/or motility in F0 and F1 males and disrupted the progression of germ cell development, as morphometric analyses exhibited an abnormal distribution of the stages of spermatogenesis in F0 males. Although plasma levels of testosterone were not affected by cannabis exposure in any ages or generations of males, dysregulated steroidogenic enzymes, Cyp11a1 and Cyp19a1, were observed in F0 testis. In the neonatal testis from F1 males, while apoptosis was not altered, DNA damage and DNMT1, but not DNMT3A and DNMT3B, were increased in germ cells following cannabis exposure. In contrast, the alterations of DNA damage and DNMT1 expression were not observed in F2 neonatal males. These results suggest that cannabis vapor exposure generationally affects male reproductive functions, probably due to disruption of spermatogenesis in the developing testis.

Induction of spermatogenesis by grafting neonatal mouse testicular tissue into epididymal fat of castrated adult mouse

Chemotherapy treatment used for childhood cancer can cause irreversible infertility in many cancer survivors. Also, it has been known that epididymal fat is necessary for spermatogenesis. In this study, spermatogenesis development was evaluated after grafting of fresh and frozen-thawed neonatal mice testicular tissue fragments to epididymal fat of bilaterally castrated adult mice. Neonatal male mice as the donor and adult male mice as the recipient were used. After bilateral castration of recipient’s mice, fresh or frozen-thawed neonatal testis tissue fragments were grafted into recipient epididymal fat. Eight weeks after implantation, grafted testicular tissue were evaluated by hematoxylin and eosin staining, real time PCR, immunofluorescence staining and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The blood of the recipient mice was collected to measure Testosterone, FSH and LH levels. Eight weeks after implantation, a gradient of different types of germ cells from spermatogonia up to the elongated spermatids were seen. The meiotic and post-meiotic genes and proteins were upregulated in both fresh and frozen grafted groups and they confirmed the meiosis and post-meiotic progression in grafted tissues. The expression of apoptosis and necrosis genes showed no significant differences between the grafted and non-grafted control groups. There were no significant differences in hormonal assessments between control and experimental groups. Epididymal fat is an area with optimal hormonal and temperature conditions, which could support spermatogenesis in grafted immature testicular tissue. This method of grafting may pave a way for fertility preservation in childhood cancer survivors.

Roles of Stra8 and Tcerg1l in retinoic acid induced spermatogonial differentiation in mouse.

Retinoic acid (RA) induces spermatogonial differentiation, but the mechanism by which it operates remains largely unknown. We developed a germ cell culture assay system to study genes involved in spermatogonial differentiation triggered by RA. Stimulated by Retinoic Acid 8 (Stra8), a RA-inducible gene, is indispensable for meiosis initiation, and its deletion results in a complete block of spermatogenesis at the pre-leptotene/zygotene stage. To interrogate the role of Stra8 in RA mediated differentiation of spermatogonia, we derived germ cell cultures from the neonatal testis of both wild type and Stra8 knock-out mice. We provide the first evidence that Stra8 plays a crucial role in modulating the responsiveness of undifferentiated spermatogonia to RA and facilitates transition to a differentiated state. Stra8-mediated differentiation is achieved through downregulation of a large portfolio of genes and pathways, most notably including genes involved in the spermatogonial stem cell self-renewal process. We also report here for the first time the role of Transcription Elongation Regulator-1 Like (Tcerg1l) as a downstream effector of RA-induced spermatogonial differentiation.

The Production of Testosterone and Gene Expression in Neonatal Testes of Rats Exposed to Diisoheptyl Phthalate During Pregnancy is Inhibited

Background: Diisoheptyl phthalate (DIHP) is a phthalate plasticizer, which is a branched phthalate. Here, we reported the effects of gestational exposure to DIHP on testis development in male rats.Methods: Pregnant Sprague-Dawley rats were orally fed with vehicle (corn oil, control) or DIHP (10, 100, 500, and 1,000 mg/kg) from gestational day (GD) 12–21. At GD21, serum testosterone levels, the number and distribution of fetal Leydig cells, and testicular mRNA and protein levels, the incidence of multinucleated gonocytes, and focal testicular hypoplasia in the neonatal testis were measured.Results: DIHP increased the fetal Leydig cell cluster size and decreased the fetal Leydig cell size with LOAEL of 10 mg/kg. DIHP did not affect the fetal Leydig cell number. DIHP significantly lowered serum testosterone levels, down-regulated the expression of steroidogenesis-related genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and testis descent-related gene (Insl3) as well as protein levels of cholesterol side-chain cleavage enzyme (CYP11A1) and insulin-like 3 (INSL3). DIHP dose-dependently increased the percentage of multinucleated gonocytes with the low observed adverse-effect level (LOAEL) of 100 mg/kg. DIHP induced focal testicular hypoplasia.Conclusion: Gestational exposure to DIHP causes testis dysgenesis in rats.

Long-term effects of acute cadmium exposure on testis immune privilege

Cadmium (Cd) is a widespread and non-biodegradable pollutant of great concern to human health. This element can affect cellular signal transduction and cell-to-cell interaction in the testis. Immune tolerance towards auto- and alloantigens is an important component of testis immunity. It is involved in spermatogenesis and hormone secretion. Plus, the immune tolerance may help to reveal the changes in testis immunity over a long period after Cd exposure. The current research was aimed at investigating the long-term effects of acute Cd exposure on testis immunity by means of elicitation of testicular immune cell composition shift induced by Cd. Cadmium chloride was intraperitoneally injected at 3 mg Cd/kg to mice. After that testis interstitial cells were stained with surface markers for leukocyte and lymphocyte subpopulations (CD45, CD11b, CD3, CD4, CD8, CD25) and analyzed cytofluorimetrically by week 4, 6, 8 and 12 after Cd administration (Cd group). To identify the delayed effects of cadmium on immune tolerance two groups of animals were subjected to intratesticular allotransplantation of neonatal testis (groups ITT and Cd+ ITT). One of the groups was administered with Cd four weeks before the transplantation (Cd+ITT group). I group served as a control that did not undergo any transplantation or Cd injection. For a better demonstration of the phenomenon of immunological tolerance of the testicles, an additional group (UKT group) was used which got grafts under the kidney capsule (non-immune privileged site).Investigation of the cell population showed that CD45+, CD11b+, CD4+, CD8+ cells were permanently present in testicular interstitial tissue in I group. Intratesticular testis transplantation increased the proportion of CD11b+ but did not have such a pronounced effect on CD8+ cells in ITT group. Moreover, the transplantation elevated CD4+ CD25+ cells known for their immunosuppressive property and promoted graft development by week 2 (histological data). Cd injection resulted in severe inflammation that quenched by week 4 (Cd and Cd+ ITT groups). This time point was chosen for transplantation in Cd+ ITT group. Such Cd pretreatment led to a high CD8+ cell proportion and to the delayed appearance of CD4+ CD25+ cells by week 2 (Cd+ ITTgroup). The finding is consistent with the impairment of graft development in Cd+ ITTgroup pretreated with Cd. Observation suggest that Cd pretreatment was associated with disproportion of interstitial immune cell populations which resulted in the impairment of immunoprotective function of the testis. The impairment of testis immunity showed itself only after several weeks of Cd administration, and only when the recipient testis immunity was provoked by alloantigens of donor testes.

Spermatogonial Type 3 Deiodinase Regulates Thyroid Hormone Target Genes in Developing Testicular Somatic Cells

Premature overexposure to thyroid hormone causes profound effects on testis growth, spermatogenesis, and male fertility. We used genetic mouse models of type 3 deiodinase (DIO3) deficiency to determine the genetic programs affected by premature thyroid hormone action and to define the role of DIO3 in regulating thyroid hormone economy in testicular cells. Gene expression profiling in the neonatal testis of DIO3-deficient mice identified 5699 differentially expressed genes. Upregulated and downregulated genes were, respectively, involved according to DAVID analysis with cell differentiation and proliferation. They included anti-Müllerian hormone and genes involved in the formation of the blood–testis barrier, which are specific to Sertoli cells (SCs). They also included steroidogenic genes, which are specific to Leydig cells. Comparison with published data sets of genes enriched in SCs and spermatogonia, and responsive to retinoic acid (RA), identified a subset of genes that were regulated similarly by RA and thyroid hormone. This subset of genes showed an expression bias, as they were downregulated when enriched in spermatogonia and upregulated when enriched in SCs. Furthermore, using a genetic approach, we found that DIO3 is not expressed in SCs, but spermatogonia-specific inactivation of DIO3 led to impaired testis growth, reduced SC number, decreased cell proliferation and, especially during neonatal development, altered gene expression specific to somatic cells. These findings indicate that spermatogonial DIO3 protects testicular cells from untimely thyroid hormone signaling and demonstrate a mechanism of cross-talk between somatic and germ cells in the neonatal testis that involves the regulation of thyroid hormone availability and action.

Prenatal Exposure to Bisphenol A, E, and S Induces Transgenerational Effects on Male Reproductive Functions in Mice

This study was performed to examine the transgenerational effects of bisphenol (BP) A analogs, BPE, and BPS on male reproductive functions using mice as a model. CD-1 mice (F0) were orally exposed to control treatment (corn oil), BPA, BPE, or BPS (0.5 or 50 µg/kg/day) from gestational day 7 (the presence of vaginal plug = 1) to birth. Mice from F1 and F2 offspring were used to generate F3 males. Prenatal exposure to BPA, BPE, and BPS decreased sperm counts and/or motility and disrupted the progression of germ cell development as morphometric analyses exhibited an abnormal distribution of the stages of spermatogenesis in F3 males. Dysregulated serum levels of estradiol-17β and testosterone, as well as expression of steroidogenic enzymes in F3 adult testis were also observed. In the neonatal testis, although apoptosis and DNA damage were not affected, mRNA levels of DNA methyltransferases, histone methyltransferases, and their associated factors were increased by BP exposure. Furthermore, BP exposure induced immunoreactive expression of DNMT3A in Sertoli cells, strengthened DNMT3B, and weakened H3K9me2 and H3K9me3 in germ cells of the neonatal testis, whereas DNMT1, H3K4me3, and H3K27ac were not affected. In adult testis, stage-specific DNMT3B was altered by BP exposure, although DNMT3A, H3K9me2, and H3K9me3 expression remained stable. These results suggest that prenatal exposure to BPA, BPE, and BPS induces transgenerational effects on male reproductive functions probably due to altered epigenetic modification following disruption of DNMTs and histone marks in the neonatal and/or adult testis.