Recovery Of Spermatogenesis Research Articles

Granulocyte colony-stimulating factor prevents loss of spermatogenesis after sterilizing busulfan chemotherapy

To determine whether granulocyte colony-stimulating factor (G-CSF) could prevent loss of spermatogenesis induced by busulfan chemotherapy via protection of undifferentiated spermatogonia, which might serve as an adjuvant approach to preserving male fertility among cancer patients. Laboratory animal study. University. Laboratory mice. Five-week-old mice were treated with a sterilizing busulfan dose and with 7 days of G-CSF or vehicle treatment and evaluated 10 weeks later (experiment 1) or 24 hours after treatment (experiment 2). Experiment 1: testis weights, epididymal sperm counts, testis histology. Experiment 2: PLZF immunofluorescent costaining with apoptotic markers. Molecular analysis of G-CSF receptor expression in undifferentiated spermatogonia. Ten weeks after treatment, busulfan-treated mice that also received treatment with G-CSF exhibited significantly better recovery of spermatogenesis and epididymal sperm counts than animals receiving busulfan alone. G-CSF led to increased numbers of PLZF+ spermatogonia 24 hours after treatment that was not accompanied by changes in apoptosis. To address the cellular target of G-CSF, mRNA for the G-CSF receptor, Csf3r, was found in adult mouse testes and cultured THY1+ (undifferentiated) spermatogonia, and cell-surface localized CSF3R was observed on 3% of cultured THY1+ spermatogonia. These results demonstrate that G-CSF protects spermatogenesis from gonadotoxic insult (busulfan) in rodents, and this may occur via direct action on CSF3R+ undifferentiated spermatogonia. G-CSF treatment might be an effective adjuvant therapy to preserve male fertility in cancer patients receiving sterilizing treatments.

Increasing testicular temperature by exposure to elevated ambient temperatures restores spermatogenesis in adult Utp14b (jsd) mutant (jsd) mice.

Because mutations in the human UTP14C gene are associated with male infertility, we sought to develop a method for fertility restoration in azoospermic mice with a mutation in the orthologous Utp14b(jsd) (jsd) gene that have spermatogonial arrest. The method is based on our observation that elevation of testicular temperatures restores spermatogonial differentiation in jsd mutant mice. To non-surgically raise intrascrotal temperatures we placed these mice in incubators at different elevated ambient temperatures. Exposure of jsd/jsd mice to ambient temperatures of 34.5 °C or 35.5 °C for 24 days increased the proportion of tubules with spermatocytes from 0% in untreated controls to over 80%. As those higher temperatures interfere with spermatid differentiation, the mice were then transferred to incubators at 32-32.5 °C for the next 24 days. These environments allowed differentiation to progress, resulting in up to 42% of tubules having late spermatids and about half of the mutant mice having spermatozoa in testicular suspensions. When these spermatozoa were used in intracytoplasmic sperm injection, all gave rise to viable healthy offspring with normal weight gain and fertility. The successful restoration of fertility in Utp14b mutant mice suggests that transient testicular warming might also be useful for spermatogenesis recovery in infertile men with UTP14C gene mutations.

Germ cell transplantation into mouse testes procedure

To illustrate the step-by-step protocol followed to assay germ cell transplantation into the seminiferous epithelium of mouse testes. Video presentation of an animal model for research in reproductive and regenerative medicine. Research laboratory. Male nude mice (NU-Foxn1(nu)). Mice were chemically sterilized with alkylant compounds (busulfan) followed by gonadal microsurgery to inject donor germ cells. Donor cells should be labeled with reporter genes, such as green fluorescent protein (GFP), lactose operon (LacZ), or alternatively design an effective strategy with specific antibodies to track them within the recipient testes. Sperm detection in the ejaculate can also be used as a read out. However, in this case detection of the donor genotype in the sperm is mandatory to elucidate their origin. In the present study we describe the complete protocol for germ cell transplant by efferent duct injection, including the preparation of recipient mice, surgery for the germ cell transplant, and analysis of recipient testes. The main strength of this technique is that it constitutes the gold standard for a functional test of the germ cell potential as only spermatogonial stem cells are able to properly colonize the seminal lumen. Both fresh and frozen/thawed testicular cells are suitable for this technique as donor germ cells. Also, enrichment of living spermatogonial stem cells, previous to the transplant, seems to improve the efficiency of colonization. For proper colonization of germ cells, the niche should be available and thus mouse strains that lack endogenous spermatogenesis such as W/W(v) mutant mice are usually used. In the case of nonmatched donor cells, seminiferous epithelium of immune-suppressed recipient mice should be germ cell depleted before the transplant. One limitation of this technique is that the procedure can take up to 3months. Also, in contrast to the full recovery of spermatogenesis in mouse-to-mouse transplants, xenotransplantation of germ cells from phylogenetically distant species, such as humans into mouse recipients, results in colonization of donor cells and spermatogonial expansion, but fail in their spermatogenic progression due to evolutive incompatibilities with the recipient niche. Xenografting of pieces of donor testis tissue under the skin of mouse hosts is an alternative approach that is currently being investigated to try to solve this limitation. Transplantation of spermatogonial stem cells into the seminal lumen of mouse testes is a functional assay that defines this cellular subpopulation by its ability to colonize it. This technique can be used as a model to elucidate the insights of spermatogonial stem cells, to produce transgenic animals by genetically manipulating donor cells before transplantation, but also it has potential applications in fertility preservation in cattle and humans as it is feasible in large animals, as recent reports have demonstrated with rhesus monkeys, that recovered spermatogenesis after allogenic transplantation, and even from human cadaver testes. Therefore spermatogonial stem cells isolated from prepuberal boys, who are treated with alkylant chemotherapy, could be returned to their testis to regenerate spermatogenesis in the future.

PAX7 expression defines germline stem cells in the adult testis.

Spermatogenesis is a complex, multistep process that maintains male fertility and is sustained by rare germline stem cells. Spermatogenic progression begins with spermatogonia, populations of which express distinct markers. The identity of the spermatogonial stem cell population in the undisturbed testis is controversial due to a lack of reliable and specific markers. Here we identified the transcription factor PAX7 as a specific marker of a rare subpopulation of A(single) spermatogonia in mice. PAX7+ cells were present in the testis at birth. Compared with the adult testis, PAX7+ cells constituted a much higher percentage of neonatal germ cells. Lineage tracing in healthy adult mice revealed that PAX7+ spermatogonia self-maintained and produced expanding clones that gave rise to mature spermatozoa. Interestingly, in mice subjected to chemotherapy and radiotherapy, both of which damage the vast majority of germ cells and can result in sterility, PAX7+ spermatogonia selectively survived, and their subsequent expansion contributed to the recovery of spermatogenesis. Finally, PAX7+ spermatogonia were present in the testes of a diverse set of mammals. Our data indicate that the PAX7+ subset of A(single) spermatogonia functions as robust testis stem cells that maintain fertility in normal spermatogenesis in healthy mice and mediate recovery after severe germline injury, such as occurs after cancer therapy.

Potential spermatogenesis recovery with bone marrow mesenchymal stem cells in an azoospermic rat model.

Non-obstructive azoospermia is the most challenging type of male infertility. Stem cell based therapy provides the potential to enhance the recovery of spermatogenesis following cancer therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the potential to differentiate or trans-differentiate into multi-lineage cells, secrete paracrine factors to recruit the resident stem cells to participate in tissue regeneration, or fuse with the local cells in the affected region. In this study, we tested whether spermatogenically-induced BMSCs can restore spermatogenesis after administration of an anticancer drug. Allogeneic BMSCs were co-cultured in conditioned media derived from cultured testicular Sertoli cells in vitro, and then induced stem cells were transplanted into the seminiferous tubules of a busulfan-induced azoospermatic rat model for 8 weeks. The in vitro induced BMSCs exhibited specific spermatogonic gene and protein markers, and after implantation the donor cells survived and located at the basement membranes of the recipient seminiferous tubules, in accordance with what are considered the unique biological characteristics of spermatogenic stem cells. Molecular markers of spermatogonial stem cells and spermatogonia (Vasa, Stella, SMAD1, Dazl, GCNF, HSP90α, integrinβ1, and c-kit) were expressed in the recipient testis tissue. No tumor mass, immune response, or inflammatory reaction developed. In conclusion, BMSCs might provide the potential to trans-differentiate into spermatogenic-like-cells, enhancing endogenous fertility recovery. The present study indicates that BMSCs might offer alternative treatment for the patients with azoospermatic infertility after cancer chemotherapy.

Adult testicular volume predicts spermatogenetic recovery after allogeneic HSCT in childhood and adolescence.

Testicular dysfunction and infertility are of major concern in long-term survivors after allogeneic hematopoietic stem cell transplantation (HSCT). This study assesses predictive factors for very long-term testicular recovery after allogeneic HSCT in childhood and adolescence. Testicular volume, sperm production and long-term need of testosterone substitution were evaluated among 106 male survivors transplanted at Huddinge and Helsinki University Hospitals from 1978 through 2000, at a mean age of 8 ± 4.6 years (range 1-17). A mean ± SD of 13 ± 4.8 years (range 4-28) had elapsed since their HSCT and the mean age of the participants was 22 ± 6.0 years (range 12-42). An adult testicular volume was recorded in 74 patients at a mean age of 19 ± 3.3 years (range 14-36). Recipients conditioned with busulfan-based regimens or regimens containing only cyclophosphamide had significantly larger adult testicular volumes (mean volume 18 ml and 16 ml vs. 9 ml, P < 0.00001, respectively) and lower serum levels of FSH (mean 9 IU and 5 IU vs. 19 IU, P < 0.01 and 0.001, respectively) compared to those conditioned with total body irradiation (TBI). Multivariate analysis demonstrated that a non-leukemia diagnosis (P < 0.01) and adult testicular volume ≥ 15 ml (P < 0.03) positively impacted spermatogenetic recovery. A larger adult testicular volume, normal serum levels of FSH and spermatozoa detected in a majority of seminal fluids after busulfan-based or cyclophosphamide conditionings suggest very long-term recovery of spermatogenesis after chemotherapy-based regimens. A simple measurement of adult testicular volume may help predict spermatogenetic potential among pediatric HSCT survivors.

Enhancement of mouse germ cell-associated genes expression by injection of human umbilical cord mesenchymal stem cells into the testis of chemical-induced azoospermic mice.

Various methods are currently under investigation to preserve fertility in males treated with high-dose chemotherapy and radiation for malignant and nonmalignant disorders. Human umbilical cord mesenchymal stem cells (HUC-MSCs), which possess potent immunosuppressive function and secrete various cytokines and growth factors, have the potential clinical applications. As a potential alternative, we investigate whether injection of HUC-MSCs into the interstitial compartment of the testes to promote spermatogenic regeneration efficiently. HUC-MSCs were isolated from different sources of umbilical cords and injected into the interstitial space of one testis from 10 busulfan-treated mice (saline and HEK293 cells injections were performed in a separate set of mice) and the other testis remained uninjected. Three weeks after MSCs injection, Relative quantitative reverse transcription polymerase chain reaction was used to identify the expression of 10 of germ cell associated, which are all related to meiosis, demonstrated higher levels of spermatogenic gene expression (2–8 fold) in HUC-MSCs injected testes compared to the contralateral uninjected testes (five mice). Protein levels for germ cell-specific genes, miwi, vasa and synaptonemal complex protein (Scp3) were also higher in MSC-treated testes compared to injected controls 3 weeks after treatment. However, no different expression was detected in saline water and HEK293 cells injection control group. We have demonstrated HUC-MSCs could affect mouse germ cell-specific genes expression. The results also provide a possibility that the transplanted HUC-MSCs may promote the recovery of spermatogenesis. This study provides further evidence for preclinical therapeutic effects of HUC-MSCs, and explores a new approach to the treatment of azoospermia.

Generation of male germ cells from mouse induced pluripotent stem cells in vitro

Germ cells are the only cell type that passes genetic information to the next generation. In most metazoan species, primordial germ cells (PGCs) were induced from epiblasts by signals from the neighboring tissues. In vitro derivation of germ cells from the pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced PSCs (iPSCs) are of great values for the treatment of infertility, for animal breeding, and for studying the mechanism of germ cell development. Although the derivations of male germ cells from PSCs have been previously reported, most of the studies failed to conduct the induction in a well-controlled and highly efficient manner. Here, we report the derivation of induced PGC-like cells (iPGCLCs) from mouse iPSCs via induced epiblast-like cells (iEpiLCs) as being monitored by the expression of enhanced green fluorescent protein gene under the control of the promoter of stimulated by retinoic acid 8 (Stra8-EGFP). The identity of iPGCLCs was characterized by examining the expression of multiple marker genes as well as by the recovery of spermatogenesis after they were transplanted to the testis of infertile W/Wv mice. Furthermore, iPGCLCs were either induced to germline stem cell-like cells (iGSCLCs) or reverted back to embryonic germ cell-like cells (iEGCLCs). In conclusion, we have established an efficient procedure for inducing iPSCs into iPGCLCs that can be further expanded and induced to more developed germ cells. This work indicates that the technology of in vitro germ cell induction is becoming more sophisticated and can be further improved.

Carnosine mitigates apoptosis and protects testicular seminiferous tubules from gamma-radiation-induced injury in mice

This study investigated the radioprotective effects of a naturally occurring dipeptide, carnosine, on testicular damage. Carnosine was administered (10, 50 and 100mgkg(-1) body weight) to male mice via intraperitoneal injection for 4days prior to gamma irradiation (2Gy). Apoptosis with the TUNEL assay and histopathological parameters were evaluated 12-h and 14-day post-irradiation. Pre-treatment with carnosine before irradiation significantly reduced the frequency of TUNEL-positive cells induced by radiation treatment at all doses by reduction factors of 1.8, 2.47 and 2.23 for carnosine at 10, 50 and 100mgkg(-1) bw, respectively, unlike that observed in the radiation alone group. Exposure to ionising radiation decreased sperm count and reduced the height and diameter of seminiferous epithelial tubules. Pre-treatment with all doses of carnosine significantly augmented seminiferous epithelial height and tubule diameter and also increased the number of germinal cells in comparison to the group treated with radiation only. These results indicate that carnosine prevents testicular dysfunction induced by gamma-irradiation via an anti-apoptotic effect; this restoration of proper testicular function ultimately leads to the recovery of spermatogenesis.

Hormone suppression with GnRH antagonist promotes spermatogenic recovery from transplanted spermatogonial stem cells in irradiated cynomolgus monkeys

Hormone suppression given before or after cytotoxic treatment stimulates the recovery of spermatogenesis from endogenous and transplanted spermatogonial stem cells (SSC) and restores fertility in rodents. To test whether the combination of hormone suppression and transplantation could enhance the recovery of spermatogenesis in primates, we irradiated (7Gy) the testes of 12 adult cynomolgus monkeys and treated six of them with gonadotropin-releasing hormone antagonist (GnRH-ant) for 8weeks. At the end of this treatment, we transfected cryopreserved testicular cells with green fluorescent protein-lentivirus and autologously transplanted them back into one of the testes. The only significant effect of GnRH-ant treatment on endogenous spermatogenesis was an increase in the percentage of tubules containing differentiated germ cells (tubule differentiation index; TDI) in the sham-transplanted testes of GnRH-ant-treated monkeys compared with radiation-only monkeys at 24weeks after irradiation. Although transplantation alone after irradiation did not significantly increase the TDI, detection of lentiviral DNA in the spermatozoa of one radiation-only monkey indicated that some transplanted cells colonized the testis. However, the combination of transplantation and GnRH-ant clearly stimulated spermatogenic recovery as evidenced by several observations in the GnRH-ant-treated monkeys receiving transplantation: (i) significant increases (~20%) in the volume and weight of the testes compared with the contralateral sham-transplanted testes and/or to the transplanted testes of the radiation-only monkeys; (ii) increases in TDI compared to the transplanted testes of radiation-only monkeys at 24weeks (9.6% vs. 2.9%; p=0.05) and 44weeks (16.5% vs. 6.1%, p=0.055); (iii) detection of lentiviral sequences in the spermatozoa or testes of five of the GnRH-ant-treated monkeys and (iv) significantly higher sperm counts than in the radiation-only monkeys. Thus hormone suppression enhances spermatogenic recovery from transplanted SSC in primates and may be a useful tool in conjunction with spermatogonial transplantation to restore fertility in men after cancer treatment.

Regeneration of Spermatogenesis after Testicular Cancer Chemotherapy

Purpose: Azoospermia is a common side effect of chemotherapy. Although most patients restore spermatogenesis over time, the exact time course has not been well described. We analyzed the recovery of spermatogenesis in testicular cancer patients following chemotherapy. Patients and Methods: 49 patients, consisting of 45 treated with a bleomycin, etoposide and cisplatin (BEP) regimen and 4 with high-dose chemotherapy, were followed up with occasional semen analyses. The primary endpoint of this study was the confirmation of motile spermatozoa in the patients' semen. Results: Among 45 patients treated with BEP, 44 recovered spermatogenesis. The recovery of spermatogenesis was delayed depending on the increase in BEP cycles. In groups of patients who received 1-2, 3 and 4 cycles, the recovery rates of spermatogenesis within 2 year were 83.3, 80.0 and 66.7%, respectively. In the group with 5-6 cycles of BEP, re-spermatogenesis was significantly delayed and no patients re-established spermatogenesis within 2 years. The patients' age and semen parameters before chemotherapy were not useful as predictive factors for the recovery of spermatogenesis. Conclusion: The recovery of spermatogenesis was rather fast and was often observed as early as several months after BEP treatment when the number of cycles was <4.

Depletion of testis cell populations in pre-pubertal Bos indicus cattle by irradiation

Recovery of spermatogenesis following a single dose of irradiation was evaluated in pre-pubertal Brahman bulls, after receiving a single dose of 3, 6, 9 or 12Gray (Gy) irradiation. Biopsy samples of testis tissue were collected and processed for immunohistology at various times following irradiation. Spermatogenic recovery was defined by the changes in tubule diameter, and absolute numbers of undifferentiated spermatogonia (PLZF positive cells) and Sertoli cells (GATA-4 positive cells) per tubule cross section. The effect of irradiation on the depletion of testicular cells was dose-dependent. Immunohistological results from both the 9 and 12Gy group showed degeneration of seminiferous tubules, compared with other doses and controls. From 2 weeks after the treatment, irradiation resulted in a significant and dramatic reduction in tubule diameter (up to 40%), number of undifferentiated spermatogonia (up to 90%) and Sertoli cells (up to 70%), which was sustained for up to 16 weeks post-irradiation in 9 and 12Gy groups (P<0.0001). However, a moderate depletion effect was observed in the 6Gy treatment groups, compared with 9 and 12Gy doses. The 6Gy treatment had significant effects on spermatogonia (up to 79% reduction) and Sertoli cell (30% reduction) numbers following irradiation (P<0.0001). In contrast, the 3Gy dose had no significant effect at either 3 or 5 weeks post-irradiation on tubule diameter, spermatogonia or Sertoli cells. In conclusion, the results from the current study suggest that treatment of recipient testes with a single dose of 6Gy irradiation can temporarily deplete spermatogonial cells in pre-pubertal Brahman bulls, whilst minimising the impact on Sertoli cells and tubule morphology.

Rad54 is required for the normal development of male and female germ cells and contributes to the maintainance of their genome integrity after genotoxic stress

Rad54 is an important factor in the homologous recombination pathway of DNA double-strand break repair. However, Rad54 knockout (KO) mice do not exhibit overt phenotypes at adulthood, even when exposed to radiation. In this study, we show that in Rad54 KO mouse the germline is actually altered. Compared with the wild-type (WT) animals, these mice have less premeiotic germ cells. This germ cell loss is found as early as in E11.5 embryos, suggesting an early failure during mutant primordial germ cells development. Both testicular and ovarian KO germ cells exhibited high radiation sensitivity leading to a long-term gametogenesis defect at adulthood. The KO female germline was particularly affected displaying decreased litter size or sterility. Spermatogenesis recovery after irradiation was slower and incomplete in Rad54 KO mice compared with that of WT mice, suggesting that loss of germ stem cell precursors is not fully compensated along the successive rounds of spermatogenesis. Finally, spermatogenesis recovery after postnatal irradiation is in part regulated by glial-cell-line-derived neurotrophic factor (GDNF) in KO but not in irradiated WT mice, suggesting that Sertoli cell GDNF production is stimulated upon substantial germ cell loss only. Our findings suggest that Rad54 has a key function in maintaining genomic integrity of the developing germ cells.

Altered protein prenylation in Sertoli cells is associated with adult infertility resulting from childhood mumps infection

Mumps commonly affects children 5-9 yr of age, and can lead to permanent adult sterility in certain cases. However, the etiology of this long-term effect remains unclear. Mumps infection results in progressive degeneration of the seminiferous epithelium and, occasionally, Sertoli cell-only syndrome. Thus, the remaining Sertoli cells may be critical to spermatogenesis recovery after orchitis healing. Here, we report that the protein farnesylation/geranylgeranylation balance is critical for patients' fertility. The expression of geranylgeranyl diphosphate synthase 1 (GGPPS) was decreased due to elevated promoter methylation in the testes of infertile patients with mumps infection history. When we deleted GGPPS in mouse Sertoli cells, these cells remained intact, whereas the adjacent spermatogonia significantly decreased after the fifth postnatal day. The proinflammatory MAPK and NF-κB signaling pathways were constitutively activated in GGPPS(-/-) Sertoli cells due to the enhanced farnesylation of H-Ras. GGPPS(-/-) Sertoli cells secreted an array of cytokines to stimulate spermatogonia apoptosis, and chemokines to induce macrophage invasion into the seminiferous tubules. Invaded macrophages further blocked spermatogonia development, resulting in a long-term effect through to adulthood. Notably, this defect could be rescued by GGPP administration in EMCV-challenged mice. Our results suggest a novel mechanism by which mumps infection during childhood results in adult sterility.

Extremely low-frequency magnetic fields can impair spermatogenesis recovery after reversible testicular damage induced by heat

Male infertility is often related to reproductive age couples experiencing fertility-related issues. Men may have fertility problems associated with reversible testicular damage. Considering that men have been increasingly exposed to extremely low-frequency magnetic fields generated by the production, distribution and use of electricity, this study analyzed whether 60 Hz and 1 mT magnetic field exposure may impair spermatogenesis recovery after reversible testicular damage induced by heat shock using rats as an experimental model. Adult male rats were subjected to a single testicular heat shock (HS, 43 °C for 12 min) and then exposed to the magnetic field for 15, 30 and 60 d after HS. Magnetic field exposure during the spermatogenesis recovery induced changes in testis components volume, cell ultrastructure and histomorphometrical parameters. Control animals had a reestablished and active spermatogenesis at 60 d after heat shock, while animals exposed to magnetic field still showed extensive testicular degeneration. Magnetic field exposure did not change the plasma testosterone. In conclusion, extremely low-frequency magnetic field may be harmful to fertility recovery in males affected by reversible testicular damage.

Spermatogenesis recovery in protein-restricted rats subjected to a normal protein diet after weaning

This study investigated the pre- and postnatal effects of protein restriction (8% vs 20% crude protein) on different parameters of spermatogenesis in adult rat offspring. Body and testis weights as well as the seminiferous tubular diameter were reduced in those animals that received the protein-restricted diet after weaning, although these parameters recovered when a 20% protein diet was offered subsequently. The numbers of spermatogonia, spermatocytes, spermatids and Leydig cells were reduced in undernourished animals, whilst the Sertoli cell number did not change. Prenatal programming effect was observed only in the spermatogonial or proliferative phase of spermatogenesis. However, the intake of the normal protein diet after weaning brought many of the testicular parameters evaluated back to normal in 70-day-old rats. A significant reduction of the meiotic index, Sertoli cell supporting capacity and spermatogenic efficiency was observed in animals subjected to protein undernutrition throughout their lives. The data presented show that protein restriction impairs the normal development of the testis in different ways, depending on the period during which the restriction was imposed, and the negative effects on spermatogenesis are more severe when undernutrition occurs from conception to adulthood; however, the return to a normal protein diet after weaning recovers the spermatogenic process.

Exogenous testosterone: a preventable cause of male infertility.

Main problemTestosterone replacement therapy inhibits spermatogenesis, representing a problem for hypogonadal men of reproductive age.MethodsA literature review of PubMed from 1990-2013. Semen analysis and pregnancy outcomes, time to recovery of spermatogenesis, serum and intratesticular testosterone levels were examined.ResultsExogenous testosterone suppresses intratesticular testosterone production, which is an absolute prerequisite for normal spermatogenesis. Therapies that protect the testis involve human chorionic gonadotropin (hCG) therapy or selective estrogen receptor modulators (SERMs), but may also include low dose hCG with exogenous testosterone. SERMs, such as clomiphene citrate, are effective for maintaining testosterone production and represent a well-tolerated, oral therapy. Routine use of aromatase inhibitors is not recommended based on a lack of long-term data.ConclusionsExogenous testosterone supplementation decreases sperm production. Studies of hormonal contraception indicate that most men have a return of normal sperm production within 1 year after discontinuation. Clomiphene citrate is a safe and effective therapy for men who desire to maintain future potential fertility. Although less frequently used in the general population, hCG therapy with or without testosterone supplementation represents an alternative treatment.

Mucuna pruriens and Its Major Constituent L-DOPA Recover Spermatogenic Loss by Combating ROS, Loss of Mitochondrial Membrane Potential and Apoptosis

BackgroundThe Ayurvedic medicinal system claims Mucuna pruriens (MP) to possess pro-male fertility, aphrodisiac and adaptogenic properties. Some scientific evidence also supports its pro-male fertility properties; however, the mechanism of its action is not yet clear. The present study aimed at demonstrating spermatogenic restorative efficacy of MP and its major constituent L-DOPA (LD), and finding the possible mechanism of action thereof in a rat model.Methodology/FindingsEthinyl estradiol (EE) was administered at a rate of 3 mg/kg body weight (BW)/day for a period of 14 days to generate a rat model with compromised spermatogenesis. MP and LD were administered in two separate groups of these animals starting 15th day for a period of 56 days, and the results were compared with an auto-recovery (AR) group. Sperm count and motility, testis histo-architecture, level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, peripheral hormone levels and testicular germ cell populations were analysed, in all experimental groups. We observed efficient and quick recovery of spermatogenesis in MP and LD groups in comparison to the auto-recovery group. The treatment regulated ROS level, apoptosis, and mitochondrial membrane potential (MMP), recovered the hypothalamic-pituitary-gonadal axis and the number of testicular germ cells, ultimately leading to increased sperm count and motility.Conclusion/Significance M. pruriens efficiently recovers the spermatogenic loss induced due to EE administration. The recovery is mediated by reduction in ROS level, restoration of MMP, regulation of apoptosis and eventual increase in the number of germ cells and regulation of apoptosis. The present study simplified the complexity of mechanism involved and provided meaningful insights into MP/LD mediated correction of spermatogenic impairment caused by estrogens exposure. This is the first study demonstrating that L-DOPA largely accounts for pro-spermatogenic properties of M. pruriens. The manuscript bears CDRI communication number 8374.

Effect of genistein administration on the recovery of spermatogenesis in the busulfan-treated rat testis

ObjectiveImpairment of spermatogenesis has been identified as an inevitable side effect of cancer treatment. Although estrogen treatment stimulates spermatogenic recovery from the impaired spermatogenesis by suppressing the intra-testicular testosterone (ITT) level, side effects of estrogen are still major impediments to its clinical application in humans. Soybeans are rich in genistein, which is a phytoestrogen that binds to estrogen receptors and has an estrogenic effect. We investigated the effects of genistein administration on ITT levels, testis weight, and recovery of spermatogenesis in rats treated with a chemotherapeutic agent, busulfan.MethodsBusulfan was administered intraperitoneally to rats, and then a GnRH agonist was injected subcutaneously into the back, or genistein was administered orally.ResultsThe weight of the testes was significantly reduced by the treatment with busulfan. The testis weight was partially restored after busulfan treatment by additional treatment with either the GnRH agonist or genistein. Busulfan also induced atrophy of a high percentage of the seminiferous tubules, but this percentage was decreased by additional treatment with either the GnRH agonist or genistein. Treatment with genistein was effective at suppressing and maintaining ITT levels comparable to that in the GnRH agonist group.ConclusionGenistein effectively suppressed ITT levels and stimulated the recovery of spermatogenesis in rats treated with a chemotherapeutic drug. This suggests that genistein may be a substitute for estrogens, for helping humans to recover fertility after cancer therapy without the risk of side effects.

Recovery of Fertility in Azoospermia Rats after Injection of Adipose-Tissue-Derived Mesenchymal Stem Cells: The Sperm Generation

The recent reports on the treatment of azoospermia patients, in which spermatozoa could not be traced in their testes, are focused more on the potential use of adult stem cells, like mesenchymal stem cells (MSCs). The aim of this study was to demonstrate the potential use of MSCs derived from adipose tissue in the treatment of azoospermia using rat disease models. After busulfan application, the rats (n = 20) were injected with the GFP+ MSCs into left rete testes. After 12 weeks, the testes with cell injection (right testes) were compared to control (left testes) after dimensional and immunohistochemical analyses. Testes treated with MSCs appeared morphologically normal, but they were atrophic in rats without stem cell treatment, in which the seminiferous tubules were empty. Spermatogenesis was detected, not in every but in some tubules of cell-treated testes. GFP+/VASA+ and GFP+/SCP1+ cells in testes indicated the transdifferentiation of MSCs into spermatogenetic cells in the appropriate microenvironment. Rats with cell treatment were mated to show the full recovery of spermatogenesis, and continuous generations were obtained. The expression of GFP was detected in the mesenchymal stem cells derived from adipose tissue and bone marrow and also in the sperms of offspring. In conclusion, MSCs might be studied for the same purpose in humans in future.