tretinoin and Infertility--Male

Topics: Germ Cells; Humans; Infertility, Male; Male; Observational Studies as Topic; PubMed; Research Report; Spermatogenesis; Spermatozoa; Tretinoin

Spermatogonia migrate to the microenvironment during the establishment from gonocytes and leave it when they differentiate. However, the mechanisms underlying the regulation of spermatogonial differentiation-associated migration remain mostly unknown. In this study, we show that spermatogonial differentiation induced by retinoic acid (RA) was accompanied with increased migration ability and elevated expression of connective tissue growth factor (CTGF), a member of the CCN family. CTGF was mainly expressed in the testicular somatic cells and committed spermatogonial progenitors. Recombinant CTGF (rCTGF) promoted the spermatogonial migration and silencing of endogenous CTGF suppressed the migration of homogenous spermatogonial cell lines. Moreover, depletion of CTGF by neutralizing antibody inhibited the elevated migration ability induced by RA, suggesting both the paracrine and autocrine roles of CTGF in spermatogonial migration associated with differentiation. Finally, CTGF interacted with β1-integrin and regulated its level in spermatogonial cell lines. Together, our study provides novel insights into the regulation of spermatogonial migration by CTGF, which may shed light on the diagnosis and treatment of male infertility.

Topics: Animals; Cell Differentiation; Cell Line; Cell Movement; Connective Tissue Growth Factor; Humans; Infertility, Male; Integrin beta1; Male; Mice; Spermatogonia; Tretinoin

Bone marrow mesenchymal stem cells (BM-MSCs) were first cultured under induction of retinoic acid (RA), Sertoli cells conditioned medium and RA + con (conditioned medium) as treatment groups. The presence of Sertoli cells was confirmed by immunocytochemistry of follicle-stimulating hormone receptor in Sertoli cells and flow cytometry by anti-Gata4 antibody. Cell viability and morphology of nucleus and cytoplasm of BM-MSCs were evaluated by MTT test and DAPI staining respectively. The expression of Oct4, Plzf, Scp3, Caspases 8, 9 and 3 genes was evaluated by RT-PCR. For increasing the accuracy of experiment, the expression of Vasa and SCP3 genes was investigated quantitatively by real-time PCR after 0, 5, 10, 15 days of culture. The results showed that the number of apoptotic cells increased in RA group. The expression of apoptosis genes (Caspases 3, 8 and 9) was also observed in this group all days of culture. Measurement of Vasa and Scp3 genes by RT-PCR confirmed the positive effects of retinoic acid on increasing of genes expression. So, in this study, a group with maximum expression of differentiation genes and minimum expression of apoptotic genes was RA + conditioned medium group. DNA fragmentation was not observed in all groups.

Topics: Animals; Bone Marrow Cells; Cell Culture Techniques; Cell Differentiation; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Flow Cytometry; Germ Cells; Infertility, Male; Male; Mesenchymal Stem Cells; Mice; Sertoli Cells; Tretinoin

The mechanisms by which varicocele affects fertility remain undetermined. Vitamin A (all-trans retinoic acid [ATRA]) is required for fertility and normal spermatogenesis; however, the mechanisms driving its action are not defined yet. Previously, we demonstrated in varicocele sperm a reduced RARα expression and that ATRA influence sperm performance. To further define vitamin A significance in male gamete and in the physiopathology of varicocele, we tested for the first time ATRA action on human sperm metabolism and antioxidant defense systems. Evaluating triglycerides content and lipase activity, in normal sperm ATRA had a lipid lowering effect, which was not observed in varicocele sperm. The modulation of the glucose-6-phosphate dehydrogenase activity, concomitantly with a reduction of the glucose content, highlight an ATRA role on glucose metabolism. ATRA induced the superoxide dismutase (SOD) and glutathione transferase activities, while it reduced the malondialdehyde and reactive oxygen species (ROS) production both in healthy and varicocele sperm. Interestingly, SOD1 and SOD2 have been localized in the acrosome and midpiece, glutathione- S-transferase omega 2 (GSTO2) in the acrosome, equatorial, and subacrosomial regions. SOD1, SOD2, and GSTO2 levels were significantly lower in varicocele with respect to healthy sperm. Herein, we discovered that ATRA treatment was able to reprogram sperm metabolism toward that of the capacitation status. The retinol protected human sperm from ROS damage enhancing the antioxidant enzymes activity, providing evidence toward the efficacy of vitamin A as therapeutic tool in improving sperm quality. These novel findings further confirm the importance of vitamin A in male fertility adding new insights into the retinoids complex biological framework.

Topics: Antioxidants; Glucose; Glucosephosphate Dehydrogenase; Glutathione Transferase; Humans; Infertility, Male; Lipase; Male; Malondialdehyde; Oxidative Stress; Reactive Oxygen Species; Spermatozoa; Superoxide Dismutase; Tretinoin; Triglycerides; Varicocele

The blood testis-barrier (BTB) is essential for maintaining homeostasis in the seminiferous epithelium. Although many studies have reported that vitamin A (VA) is required for the maintenance of spermatogenesis, the relationships between the BTB, spermatogenesis and VA have not been elucidated. In this study, we analyzed BTB assembly and spermatogenesis in the testes of mice fed the VA-deficient (VAD) diet from the prepubertal period to adulthood. During the prepubertal period, no changes were observed in the initiation and progression of the first spermatogenic wave in mice fed the VAD diet. However, the numbers of preleptotene/leptotene spermatocytes derived from the second spermatogenic wave onwards were decreased, and initial BTB formation was also delayed, as evidenced by the decreased expression of mRNAs encoding BTB components and VA signaling molecules. From 60 days postpartum, mice fed the VAD diet exhibited apoptosis of germ cells, arrest of meiosis, disruption of the BTB, and dramatically decreased testis size. Furthermore, vacuolization and calcification were observed in the seminiferous epithelium of adult mice fed the VAD diet. Re-initiation of spermatogenesis by VA replenishment in adult mice fed the VAD diet rescued BTB assembly after when the second spermatogenic wave initiated from the arrested spermatogonia reached the preleptotene/leptotene spermatocytes. These results suggested that BTB integrity was regulated by VA metabolism with meiotic progression and that the impermeable BTB was required for persistent spermatogenesis rather than meiotic initiation. In conclusion, consumption of the VAD diet led to critical defects in spermatogenesis progression and altered the dynamics of BTB assembly.

Topics: Animals; Apoptosis; Biomarkers; Blood-Testis Barrier; Calcinosis; Diet; Disease Models, Animal; Epididymis; Female; Gene Expression Regulation, Developmental; Infertility, Male; Male; Metaplasia; Mice; Mice, Inbred C57BL; Organ Size; Spermatogenesis; Spermatogonia; Testis; Tretinoin; Vacuoles; Vitamin A Deficiency

Historically, our understanding of molecular genetic aspects of germ cell development has been limited. Recently, results demonstrated that the derivation of pluripotent stem cells may provide the necessary genetic system to study germ cell development. Here, we characterized an induced pluripotent stem cell (iPSC) line, which can spontaneously differentiate into embryonic bodies (EBs) after 3 days of suspension culture, expressing specific markers of three germ layers. Then, we induced the iPSCs to differentiate into germ cells by culturing adherent EBs in retinoic acid (RA) and porcine follicular fluid (PFF) differentiation medium or seminiferous tubule transplantation. Our results indicated that RA and PFF were beneficial for the derivation of germ cells and oocyte-like cells from iPSCs, and iPSCs transplantation could make a contribution to repairing the testis of infertile mice. Our study offers an approach for further study on the development and the differentiation of germ cells derived from iPSCs.

Topics: Animals; Busulfan; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Culture Media; Embryoid Bodies; Female; Follicular Fluid; Gene Expression Profiling; Germ Cells; Induced Pluripotent Stem Cells; Infertility, Male; Male; Mice; Seminiferous Tubules; Suspensions; Swine; Teratoma; Transplantation, Heterotopic; Tretinoin

Previous studies have demonstrated that mouse- and human-induced pluripotent stem (iPS) cells can differentiate into primordial germ cells in vitro. However, up to now it is not known whether iPS cells would be able to differentiate into male germ cells in vivo. The aim of this study was to explore differentiation potential of iPS cells to male germ cells in vitro and in vivo.. In this study, approaches using in vitro retinoic acid induction and in vivo ectopic transplantation were combined to induce iPS cells to become male germ cells.. RT-PCR showed that expression of pre-meiotic and meiotic germ cell-specific genes was enhanced in iPS cell-derived embryoid bodies (EBs) compared to mRNA transcripts of iPS cells. Immunofluorescence analysis revealed that iPS cell-derived EBs positively expressed germ-cell markers VASA, c-Kit and SCP3. Furthermore, iPS cell-derived cells dissociated from EBs were injected with testicular cells into dorsal skin of mice. Histological examination showed that iPS cell-derived cells could reconstitute seminiferous tubules, and meanwhile, iPS cell-derived germ cells could settle at basement membranes of reconstituted tubules.. Our results suggest that iPS cells are able to differentiate into male germ cells in vitro and that reconstituted seminiferous tubules may provide a functional niche for exogenous iPS cell-derived male germ cells. Derivation of male germ cells from iPS cells has potential application for treating male infertility and provides an ideal platform for elucidating molecular mechanisms of male germ-cell development.

Topics: Animals; Base Sequence; Cell Differentiation; DNA Primers; Embryoid Bodies; Green Fluorescent Proteins; Humans; In Vitro Techniques; Induced Pluripotent Stem Cells; Infertility, Male; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Nude; Mice, Transgenic; RNA, Messenger; Seminiferous Tubules; Spermatogenesis; Transplantation, Heterologous; Tretinoin