A comparison of the effects of fetal bovine serum and newborn calf serum on cell growth and maintenance of cryopreserved mouse spermatogonial stem cells.

Serum is a common supplement that is widely used to protect various cells and tissues from cryopreservation because it provides the necessary active components for cell growth and maintenance. In this study, we compared the effects of newborn calf serum (NCS) and fetal bovine serum (FBS) on the cryopreservation of mouse spermatogonial stem cells (SSCs). The isolated SSCs were cryopreserved in two groups: freezing medium that contained 10% DMSO (dimethyl sulfoxide) and 10% FBS in DMEM (Dulbecco's Modified Eagle's Medium) (group 1) and freezing medium that contained 10% DMSO and 10% NCS in DMEM (group 2). Real-time PCR was performed for stemness gene expression. The SSCs' viability was performed by trypan blue. We observed that the SSCs had increased viability in the NCS-freeze/thaw group (87.82%) compared to the FBS-freeze/thaw group (79.83%), but this increase was not statistically significant (P < 0.105). Promyelocytic leukemia zinc finger (Plzf) and Lin28 gene expression levels in the NCS-frozen/thawed SSCs were not significantly different compared to the FBS-frozen/thawed SSCs; however, Nanog gene expression increased considerably, and Dazl gene expression decreased significantly. The results in this study demonstrated that the presence of NCS in a solution of cryopreserved SSCs increased their viability after freeze/thawing and might promote the proliferation of cultivated SSCs in vitro by increasing the relative expression of Nanog.

Ethanolic extract of Moringa oleifera leaves alleviate cyclophosphamide-induced testicular toxicity by improving endocrine function and modulating cell specific gene expression in mouse testis.

ETHNOPHARMACOLOGICAL RELEVANCE: Moringa oleifera Lam. is known for its nutritional and ethno medicinal values due to the presence of wide array of phytochemicals with multiple biological activities. We have previously reported that ethanolic extract of Moringa oleifera leaves (MOE) ameliorated cyclophosphamide (CP)-induced testicular toxicity and improved functional integrity of spermatozoa as well as spermatogenic cells. AIM OF THE STUDY: The present study was planned to investigate whether the mitigation of CP-induced testicular toxicity by MOE is mediated via modulation of endocrine profile, genes associated with function of different cell types and enhancement of DNA repair response in spermatogonial cells. MATERIALS AND METHODS: Adult Swiss albino mice (8 week) were injected with CP (100 mg/kg, one dose in a week for 3 weeks) and MOE (100 mg/kg, 5 doses in a week for 4 weeks) either alone or in combination intraperitoneally. At 35 day post CP injection (first dose), the functional characteristics such as count, motility, head morphology and DNA integrity were assessed in epididymal spermatozoa. Key reproductive hormones like testosterone, follicle stimulating hormone (FSH) and Inhibin B concentration were analyzed in serum and testis. In addition, mRNA expression of genes pertaining to the function of Leydig, Sertoli and spermatogonial cells as well as antioxidant enzymes were evaluated in the testis. To understand the DNA damage and repair process in germ cells, prepubertal (2 week) mice were administered with single dose of CP (200 mg/kg) and/or MOE (100 mg/kg) and analyzed for expression of DNA damage (γ-H2AX, P53 and Caspase3) and repair genes (Rad51 and Ku80) in isolated spermatogonial cells at various time points after treatment. RESULTS: CP administration resulted in decrease in count, motility and increase in morphological defects and DNA damage in spermatozoa. Testosterone level was marginally decreased while there was a significant increase in FSH (p < 0.001) and decrease in inhibin B (p < 0.05) observed in CP treated mice. Administration of MOE prior to CP, improved sperm functional characteristics, decreased FSH and increased inhibin B levels. Expression of Abp was down-regulated while Transferrin, Fshr and Gata4 (Sertoli cell specific genes) were up-regulated in testis treated with CP. Administration of CP down-regulated the expression of Oct4 and Ddx4 (Spermatogonia specific genes). MOE administration was shown to ameliorate CP-induced damage by modulating the expression of genes specific to Sertoli and spermatogenic cells. Furthermore, MOE treatment reduced CP-induced DNA damage as evident from lower percentage of γ-H2AX positive spermatogonial cells. CONCLUSION: Administration of MOE mitigated CP-induced testicular damage by improving blood and, intra-testicular hormonal milieu as well as modulating the expression of genes pertaining to Sertoli and spermatogonial cells.

Regulation of spermatogonial development by Fsh: The complementary roles of locally produced Igf and Wnt signaling molecules in adult zebrafish testis.

Spermatogenesis is a cellular developmental process characterized by the coordinated proliferation and differentiation activities of somatic and germ cells in order to produce a large number of spermatozoa, the cellular basis of male fertility. Somatic cells in the testis, such as Leydig, peritubular myoid and Sertoli cells, provide structural and metabolic support and contribute to the regulatory microenvironment required for proper germ cell survival and development. The pituitary follicle-stimulating hormone (Fsh) is a major endocrine regulator of vertebrate spermatogenesis, targeting somatic cell functions in the testes. In fish, Fsh regulates Leydig and Sertoli cell functions, such as sex steroid and growth factor production, processes that also control the development of spermatogonia, the germ cell stages at the basis of the spermatogenic process. Here, we summarize recent advances in our understanding of mechanisms used by Fsh to regulate the development of spermatogonia. This involves discussing the roles of insulin-like growth factor (Igf) 3 and canonical and non-canonical Wnt signaling pathways. We will also discuss how these locally active regulatory systems interact to maintain testis tissue homeostasis.

Functional anatomy of the male reproductive system of the American lobster (Homarus americanus).

Despite supporting a valuable fishery, the reproductive system of the male American lobster (Homarus americanus) is poorly understood. The elongated H-shaped testis is responsible for spermatogenesis and is composed of follicles, a common collecting duct with interlaced scattered striated muscles, and a serosa as an external wall. Sertoli cells are associated with the spermatogenesis that produces spermatozoa, which are transferred to the collecting duct through a temporary passageway. Spermatogenesis is asynchronous between follicles and occurs on a continuous basis. The anterior and posterior lobes of the testes are independent and connect to the vasa deferentia through the Y-shaped collecting tubules that have a different cell anatomy and function than the two organs they connect. The vas deferens is divided into four regions. Spermatophores, produced in the proximal vas deferens, are packets of spermatozoa encapsulated in a single layer-the spermatophoric wall, which is composed of mucopolysaccharide acid. Large dense ovoid granules and the seminal fluid, composed of acidic sulfated mucosubstances, are secreted in the median vas deferens. Spermatophores within these secreted substances (i.e., semen) are stored in the distal vas deferens that, with the spermiduct (last region of the vas deferens), is responsible for the extrusion of the semen by striated muscle contractions. Smooth muscles suggest a peristaltic movement of the spermatophores within the vas deferens. Finally, the gonopores and the first pair of pleopods (i.e., gonopod) move the semen to the female seminal receptacle during copulation.

Antiapoptotic effects of vitamins C and E against cypermethrin-induced oxidative stress and spermatogonial germ cell apoptosis.

Toxicological studies have demonstrated the relation between use of agrochemicals and fertility issues within males. Thus, the present study aimed to elucidate the propensity of cypermethrin (CYP) in bringing testicular germ cell apoptosis and effective attenuation by vitamins C and E in caprines. Reproductive toxicity of CYP was evaluated using histomorphological, cytological, and biochemical changes in the testicular germ cells in dose-dependent (1, 5, 10 µg/mL) and time-dependent (4, 6, 8 h) manner. Histological and ethidium bromide/acridine orange fluorescence staining exhibited that vitamins C and E (0.5 and 1.0 mM) successfully diminished the CYP-induced testicular germ cells apoptosis. CYP exposure along with vitamins C and E supplementation also resulted in significantly increased ferric reducing antioxidant power activity along with the antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione-s-transferase, and decreased lipid peroxidation in testicular germ cells. Thus, vitamins C and E ameliorated CYP-induced testicular germ cell apoptosis, thereby preventing spermatogonial cells degeneration and male infertility.

Berberine reinforces Sertoli cells niche and accelerates spermatogonial stem cells renewal in experimentally-induced varicocele condition in rats.

BACKGROUND: Varicocele is present in 10-20% of the male infertile population. PURPOSE: Present study was done to demonstrate the reinforcing effect of berberine (BBR), as an antioxidant and anti-inflammatory agent, on Sertoli cells-related niche and spermatogonial stem cells (SSCs) self-renewal in experimentally-induced VCL condition. STUDY DESIGN: 50 mature male Wistar rats were divided into control, control-sham, non-treated VCL-induced, 50 mg kg-1 and 100 mg kg-1 BBR-treated VCL-induced groups. METHODS: The Leydig and Sertoli cells distribution and Leydig cells steroidogenic activity, expression of glial cell line-derived neurotrophic factor (GDNF), proto-oncogene Rearranged during Transfection (c-RET) receptor, Ets variant gene 5 (Etv5) and B-cell chronic lymphocytic leukemia (CLL)/lymphoma 6, member B (Bcl-6b) at mRNA and protein levels were analyzed. The mRNA integrity and DNA fragmentation were assessed. Finally, the serum levels of testosterone, inhibin B and testicular total antioxidant capacity, total thiol molecules, catalase, and malondialdehyde were evaluated. RESULTS: Observations revealed that, the BBR significantly enhanced VCL-reduced Leydig and Sertoli cells population, maintained Leydig-Sertoli cells network, enhanced GDNF, c-RET Etv5 and Bcl6b expression, up-regulated testicular antioxidant and endocrine status. CONCLUSION: The BBR by boosting Leydig-Sertoli cells network up-regulates the GDNF, Etv5 and Bcl-6b expression/synthesis in SSCs, which in turn improves SSCs self-renewal activities. Thus, the BBR could be considered as an appropriate agent for antioxidant therapy of VCLs. However, more studies with bigger sample number and focus on BBR-induced effects on other genes involving in the self-renewal process are needed to have more deterministic results.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.

The effects of melatonin on colonization of neonate spermatogonial mouse stem cells in a three-dimensional soft agar culture system.

BACKGROUND: Melatonin is a pleiotropic hormone with powerful antioxidant activity both in vivo and in vitro. The present study aimed to investigate the effects of melatonin on the proliferation efficiency of neonatal mouse spermatogonial stem cells (SSCs) using a three-dimensional soft agar culture system (SACS) which has the capacity to induce development of SSCs similar to in vivo conditions. METHODS: SSCs were isolated from testes of neonate mice and their purities were assessed by flow cytometry using PLZF antibody. Isolated testicular cells were cultured in the upper layer of the SACS in αMEM medium in the absence or presence of melatonin extract for 4 weeks. RESULTS: The identity of colonies was confirmed by alkaline phosphatase staining and immunocytochemistry using PLZF and α6 integrin antibodies. The number and diameter of colonies of SSCs in the upper layer were evaluated at days 14 and 28 of culture. The number and diameter of colonies of SSCs were significantly higher in the melatonin group compared with the control group. The levels of expression of ID-4 and Plzf, unlike c-kit, were significantly higher in the melatonin group than in the control group. CONCLUSIONS: Results of the present study show that supplementation of the culture medium (SACS) with 100 µM melatonin significantly decreased reactive oxygen species (ROS) production in the treated group compared with the control group, and increased SSC proliferation.

In vitro effects of melatonin on colonization of neonate mouse spermatogonial stem cells.

We have recently reported that antioxidant supplements enhance the efficacy of cryopreserved spermatogonial stem cells. Melatonin is considered a free radical scavenger which has direct and indirect antioxidant effects in in vitro and in vivo microenvironments. Due to the anti-apoptotic properties of melatonin, researchers have proposed that melatonin may improve the efficiency of spermatogonial stem cell (SSC) transplantation. However, the appropriate methodology which facilitates SSC proliferation remains to be determined. Identification of a proper propagation system is essential for the future application of SSCs in the field of infertility. The aim of the present study was to investigate the effects of melatonin on the colonization of SSCs. SSCs were isolated from the testes of three to six day old mice, and their purities were assessed by cytometry using Plzf antibody. Isolated testicular cells were cultured in the absence or presence of melatonin extract for two weeks. Suppression of differentiation and maintenance of spermatogonial stem cells was confirmed by alkaline phosphatase staining and immunocytochemistry using Plzf antibody. The number and diameter of the colonies of SSCs were assessed during the 7th and 14th days of culture, and the expression of Id4, Plzf, and C-kit were evaluated using real-time PCR at the end of the culture period. The survival rate of the cultured cells in the presence of melatonin was significantly higher than the control group. The number and diameter of colonies also increased in the cells treated with melatonin. The results of our study suggest that culture of SSCs with 100 µM melatonin supplementation can increase SSCs proliferation significantly.

Yangjing Capsule extract promotes proliferation of GC-1 spg cells via up-regulated POU3F1 pathway.

As is similar to glial cell line-derived neurotrophic factor (GDNF), the Yangjing Capsule (YC) extract could also lead to proliferation of spermatogonial stem cells (SSCs) by stimulating the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway; however, the regulatory effect of YC extract on the expression of POU3F1 still remains unknown. The objective of this study is to determine whether the transcription factor POU3F1 is up-regulated by YC extract through the PI3K/AKT signaling pathway to regulate SSCs survival and proliferation. Cultured GC-1 spermatogonial (spg) cells were treated with 0.01, 0.1, and 1 mg/mL YC extract for 48 h. Cell viability was analyzed using MTT assay, while POU3F1 expression was quantitatively detected using real time-polymerase chain reaction and Western blot analysis. POU3F1, GDNF family receptor alpha1 (GFRα1) short interfering ribonucleic acid (siRNA), and LY294002 (PI3K inhibitor) were applied as blockers to explore the underlying pathway. After 48 h treatment with YC extract, GC-1 spg cells proliferated and POU3F1 expression was significantly increased in a dose-dependent manner. POU3F1 siRNA partially blocked those effects of YC extract. Both GFRα1 siRNA and LY294002, as upstream blockers, reduced POU3F1 expression induced by YC extract. The conclusion is that YC extract promotes proliferation of GC-1 spg cells via up-regulation of POU3F1. The potential mechanism is that YC extract triggers the activation of the PI3K/AKT pathway and then up-regulates POU3F1 expression.

Granulocyte colony-stimulating factor (G-CSF) promotes spermatogenic regeneration from surviving spermatogonia after high-dose alkylating chemotherapy.

BACKGROUND: The lifesaving chemotherapy and radiation treatments that allow patients to survive cancer can also result in a lifetime of side-effects, including male infertility. Infertility in male cancer survivors is thought to primarily result from killing of the spermatogonial stem cells (SSCs) responsible for producing spermatozoa since SSCs turn over slowly and are thereby sensitive to antineoplastic therapies. We previously demonstrated that the cytokine granulocyte colony-stimulating factor (G-CSF) can preserve spermatogenesis after alkylating chemotherapy (busulfan). METHODS: Male mice were treated with G-CSF or controls before and/or after sterilizing busulfan treatment and evaluated immediately or 10-19 weeks later for effects on spermatogenesis. RESULTS: We demonstrated that the protective effect of G-CSF on spermatogenesis was stable for at least 19 weeks after chemotherapy, nearly twice as long as previously shown. Further, G-CSF treatment enhanced spermatogenic measures 10 weeks after treatment in the absence of a cytotoxic insult, suggesting G-CSF acts as a mitogen in steady-state spermatogenesis. In agreement with this conclusion, G-CSF treatment for 3 days before busulfan treatment exacerbated the loss of spermatogenesis observed with G-CSF alone. Reciprocally, spermatogenic recovery was modestly enhanced in mice treated with G-CSF for 4 days after busulfan. These results suggested that G-CSF promoted spermatogonial proliferation, leading to enhanced spermatogenic regeneration from surviving SSCs. Similarly, there was a significant increase in proportion of PLZF+ undifferentiated spermatogonia that were Ki67+ (proliferating) 1 day after G-CSF treatment. CONCLUSIONS: Together, these results clarify that G-CSF protects spermatogenesis after alkylating chemotherapy by stimulating proliferation of surviving spermatogonia, and indicate it may be useful as a retrospective fertility-restoring treatment.

Differentiation of Spermatogonia Stem Cells into Functional Mature Neurons Characterized with Differential Gene Expression.

Transplantation of embryonic stem cells (ESCs) is a promising therapeutic approach for the treatment of neurodegenerative diseases. However, ESCs are not usable clinically due to immunological and ethical limitations. The identification of an alternative safe cell source opens novel options via autologous transplantation in neuro-regeneration circumventing these problems. Here, we examined the neurogenic capacity of embryonic stem-like cells (ES-like cells) derived from the testis using neural growth factor inducers and utilized them to generate functional mature neurons. The neuronal differentiation of ES-like cells is induced in three stages. Stage 1 is related to embryoid body (EB) formation. To induce neuroprogenitor cells, EBs were cultured in the presence of retinoic acid, N2 supplement and fibroblast growth factor followed by culturing in a neurobasal medium containing B27, N2 supplements for additional 10 days, to allow the maturation and development of neuronal progenitor cells. The neurogenic differentiation was confirmed by immunostaining for markers of mature neurons. The differentiated neurons were positive for Tuj1 and Tau1. Real-time PCR dates indicated the expression of Nestin and Neuro D (neuroprogenitor markers) in induced cells at the second stage of the differentiation protocol. The differentiated mature neurons exhibited the specific neuron markers Map2 and ß-tubulin. The functional maturity of neurons was confirmed by an electrophysiological analysis of passive and active neural membrane properties. These findings indicated a differentiation capacity of ES-like cells derived from the testis to functionally mature neurons, which proposes them as a novel cell source for neuroregenerative medicine.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

STUDY QUESTION: Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? SUMMARY ANSWER: We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. WHAT IS KNOWN ALREADY: Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in vitro or testosterone production was observed. LIMITATIONS, REASONS FOR CAUTION: The human testis is very different in physiology from the rat testis, further investigations are still needed to optimize the organ culture system for future use in humans. WIDER IMPLICATIONS OF THE FINDINGS: The successful differentiation of undifferentiated spermatogonia using the testis explant culture system might be employed in future to produce sperm from human spermatogonia as a clinical tool for fertility preservation in boys and men suffering infertility. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was supported financially by the Frimurare Barnhuset in Stockholm, the Paediatric Research Foundation, Jeanssons Foundation, Sällskåpet Barnåvard in Stockholm, Swedish Research Council/Academy of Finland, Emil and Wera Cornells Foundation, Samariten Foundation, the Swedish Childhood Cancer Foundation as well as through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska Institutet. All authors declare no conflicts of interests.

Transplantation of testicular tissue in alginate hydrogel loaded with VEGF nanoparticles improves spermatogonial recovery.

Transplantation of cryopreserved immature testicular tissue (ITT) is a promising strategy to restore fertility in young boys facing gonadotoxic treatments. However, up to now, limited spermatogonial recovery has been achieved in xenografting models used to evaluate the potential of cryopreserved tissue transplantation. When comparing avascular xenografts of cryopreserved and fresh human ITT into a mouse model, the number of spermatogonia was significantly reduced, regardless of the cryopreservation procedure used. To improve tissue engraftment, revascularization and hence spermatogonial survival, ITT was embedded in two types of hydrogel loaded with VEGF nanoparticles. Small pieces (±1mm(3)) of testicular tissue were grafted in NMRI mice as follows: grafted without encapsulation, grafted after encapsulation in fibrin, in alginate, in fibrin-VEGF-nanoparticle (NP) and in alginate-VEGF-NP. Non-grafted tissue served as control. After 5 and 21days of implantation, seminiferous tubule integrity, revascularization and spermatogonial recovery were evaluated by histology and immunohistochemistry. Seminiferous tubule integrity ranged from 13.3% to 39.6% and 42.7% to 68.7% on day 5 and day 21, respectively. Vascular density on day 5 was found to be higher in VEGF supplemented groups, regardless of the hydrogel used. Staining for phosphorylated VEGF receptor 2 and endothelial proliferation on day 5 was higher in all groups compared to non-grafted avascular controls. Spermatogonial recovery ranged between 14.8% and 27.3% on day 21 and was significantly higher in the alginate and alginate-VEGF-NP groups. The present study demonstrates the potential of alginate hydrogel loaded with nanoencapsulated growth factors to improve cryopreserved tissue engraftment.

Improving the Efficacy of Cryopreservation of Spermatogonia Stem Cells by Antioxidant Supplements.

Cryopreservation of spermatogonial stem cells (SSCs) is an applicable method for young males seeking fertility preservation before starting a treatment. It increases reactive oxygen species (ROS) formation and oxidative stress, which damages cellular structures. In this study, we added two antioxidants, catalase and α-tocopherol (α-TCP), to the basic freezing medium to evaluate their effects on the efficiency of SSCs. SSCs were isolated from testes of 3- to 6-day-old male mice using enzymatic digestion. The enrichment of isolated cells was evaluated by flow cytometry and Stra8 antibody. Catalase (40 µg/mL), or α-TCP (200 µg/mL) was added to the basic freezing medium. The cell viability was evaluated by the methylthiazoltetrazolium (MTT) assay. After thawing, cells were cultured for 1 month, and the expression pattern of specific genes of SSCs and the ability of the cells to restore spermatogenesis were used to determine the efficiency of the cryopreservation method. The survival rate of the frozen cells in the presence of catalase or α-TCP was significantly higher than the control group (p < 0.05). The number of colonies and their diameter measured after 1 month were significantly higher in the antioxidant groups than in the control group (p < 0.05). Gene expression and resumption of spermatogenesis also followed the same pattern. Thus, adding antioxidants to the basic freezing medium can be helpful in increasing the quality and viability of SSCs after cryopreservation. This new approach to stem cells cryopreservation can also be a promising strategy for fertility preservation in patients who suffer from malignancy.

In vitro effects of date palm (Phoenix dactylifera L.) pollen on colonization of neonate mouse spermatogonial stem cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Date palm (Phoenix dactylifera L.) pollen (DPP) is widely used as a folk remedy for male infertility treatment, and has well known medicinal effects. AIM OF THE STUDY: This study aimed to determine the in vitro effects of DPP on the efficiency of neonate mouse spermatogonial stem cells (SSCs) proliferation. MATERIAL AND METHODS: Sertoli and SSCs were isolated from 6 to 10-days-old mouse testes, and their identity was confirmed using immunocytochemistry against cytokeratin for sertoli cells and PLZF, Oct-4 and CDH-1 for SSCs. Isolated testicular cells were cultured in the absence or presence of 0.06, 0.25 and 0.62mg/ml concentrations of DPP aqueous extract for 2 weeks. The number and diameter of SSC colonies were assessed during third, 7th, 9th and 14th day of culture, and the expression of the Mvh, GFRα-1 and Oct-4 was evaluated using quantitative PCR at the end of the culture period. The significance of the data was analyzed using ANOVA and paired samples t-test and Tukey and Bonferroni test as post hoc tests at the level of p≤0.05. RESULTS: Pattern assay of colony formation showed that SSCs numbers increased in the present of 0.62mg/ml concentration of DPP extract with higher slop relative to other groups (P <0.05). Colony diameters had no significant difference between groups in 3th, 7th, 9th and 14th days after culture. The Mvh and Oct-4 genes expression had no significant difference between groups, while GFRα1 expression was increased significantly in cells treated with 0.06mg/ml concentration relative to other groups (P<0.05). CONCLUSION: It seems that co-culture of SSCs with sertoli sells in the presence of low doses of DPP can increase SSCs proliferation and keep their stemness state, while higher concentrations can differentiate the treated cells.

Factors supporting long-term culture of bovine male germ cells.

Spermatogonial stem cells (SSCs) are unipotent in nature, but mouse SSCs acquire pluripotency under the appropriate culture conditions. Although culture systems are available for rodent and human germ-cell lines, no proven culture system is yet available for livestock species. Here, we examined growth factors, matrix substrates and serum-free supplements to develop a defined system for culturing primitive germ cells (gonocytes) from neonatal bovine testis. Poly-L-lysine was a suitable substrate for selective inhibition of the growth of somatic cells and made it possible to maintain a higher gonocyte:somatic cell ratio than those maintained with gelatin, collagen or Dolichos biflorus agglutinin (DBA) substrates. Among the serum-free supplements tested in our culture medium, knockout serum replacement (KSR) supported the proliferation and survival of gonocytes better than the supplements B-27 and StemPro-SFM after sequential passages of colonies. Under our optimised culture conditions consisting of 15% KSR supplement on poly-L-lysine-coated dishes, the stem-cell and germ-cell potentials of the cultured gonocytes were maintained with normal karyotype for more than 2 months (over 13 passages). The proposed culture system, which can maintain a population of proliferating bovine germ stem cells, could be useful for studying SSC biology and germline modifications in livestock animals.

In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues.

Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sld mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.

Effects of different levels of dietary selenium on the proliferation of spermatogonial stem cells and antioxidant status in testis of roosters.

The objective of this study was to investigate the different levels of dietary Se (from sodium selenite) on the proliferation of SSCs (spermatogonial stem cells) in testis of roosters. Also, the antioxidant status and Se content in blood plasma and testis were evaluated. A total of eighty 12-week-old Hy-Line Variety white roosters at an averaged body weight of 1.38 ± 0.2 kg were selected and randomly divided into four experimental groups. They were fed with the basal diet (0.044 mgSe/kg DM) supplemented with 0 (control), 0.5, 1.0 or 2.0 mgSe/kg DM (from sodium selenite). After the feeding experiment, blood and testis samples were collected for analysis of the antioxidant status and Se concentration. The testis samples were also used to examine the Thy-1 and ß1-integrin mRNA expression by RT-PCR and detect the population of SSCs by immunofluorescence analysis. The results show that Se concentration in blood and testis of the animals was progressively increased with the increasing Se level in diet. The highest GSH-Px (glutathione peroxidase) activity and lowest MDA content in blood and testis was obtained in the treatment of 0.5mg/kg. RT-PCR analysis showed that mRNA expression of SSCs markers were significantly lower in the control and 1.0mg/kg groups when compared with that in the treatment of 0.5mg/kg. A similar trend was observed in the population of SSCs analyzed by immunofluorescence assay. These data suggest that dietary Se can influence the population of SSCs of roosters during spermatogenesis and that oxidative stress can modulate SSCs behavior through regulating some key factors during spermatogenesis.

A noninvasive method for in situ determination of mating success in female American lobsters (Homarus americanus).

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female's seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George's Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.