Different Stages Of Spermatogenesis Research Articles

Effect of IL-1β on the Development of Spermatogenesis In Vitro in Normal and Busulfan-Treated Immature Mice.

Gonadotoxic agents could impair spermatogenesis and may lead to male infertility. The present study aimed to evaluate the effect of IL-1β on the development of spermatogenesis from cells isolated from seminiferous tubules (STs) of normal and busulfan-treated immature mice in vitro. Cells were cultured in a 3D in vitro culture system for 5 weeks. We examined the development of cells from the different stages of spermatogenesis by immunofluorescence staining or qPCR analyses. Factors of Sertoli and Leydig cells were examined by qPCR analysis. We showed that busulfan (BU) treatment significantly reduced the expression of testicular IL-1β in the treated mice compared to the control group (CT). Cultures of cells from normal and busulfan-treated immature mice induced the development of pre-meiotic (Vasa), meiotic (Boule), and post-meiotic (acrosin) cells. However, the percentage of developed Boule and acrosin cells was significantly lower in cultures of busulfan-treated mice compared to normal mice. Adding IL-1β to both cultures significantly increased the percentages of Vasa, Boule, and acrosin cells compared to their controls. However, the percentage of Boule and acrosin cells was significantly lower from cultures of busulfan-treated mice that were treated with IL-1β compared to cultures treated with IL-1β from normal mice. Furthermore, addition of IL-1β to cultures from normal mice significantly increased only the expression of androgen receptor and transferrin but no other factors of Sertoli cells compared to their CT. However, the addition of IL-1β to cultures from busulfan-treated mice significantly increased only the expression of androgen-binding protein and the FSH receptor compared to their CT. Adding IL-1β to cultures of normal mice did not affect the expression of 3βHSD compared to the CT, but it significantly reduced its expression in cultures from busulfan-treated mice compared to the CT. Our findings demonstrate the development of different stages of spermatogenesis in vitro from busulfan-treated mice and that IL-1β could potentiate this development in vitro.

Biological Parameters and Spermatogenesis in Razorfish (Pelecus cultratus) Population Inhabiting the Largest Shallow Lake of Central Europe (Lake Balaton): Studies for In Vitro Conservation Purposes

The study aimed to investigate body parameters and the process of spermatogenesis from April to September. In addition, it sought to test the applicability of sperm cryopreservation for conservation purposes in a razorfish (Pelecus cultratus) population of Lake Balaton, the largest shallow lake in Central Europe. During the aforementioned period, measurements were taken for the standard length (SL, cm) and body weight (BW, g), and the sex of specimens was determined. Cells at different stages of spermatogenesis (spermatogonia-SG, spermatocytes-SC, spermatids-ST, and spermatozoa-SZ) were quantified monthly for each male sample. Sperm samples collected at the end of May were cryopreserved using a method designed for common carp. No significant differences were found between males (SL: 25 ± 2 cm and BW: 146 ± 38 g) and females (SL: 26 ± 3 cm and BW: 168 ± 53 g) in terms of measured body parameters. No significant correlations were found between the sex, SL, and BW. High standard deviations were observed for all mean values in all sampling periods, possibly due to the low number of individual samples per month. A significantly higher proportion of SC compared to SZ was found in April. In May, no differences were observed between the four groups. Significantly more SG and SZ than SC and ST were observed in June. In August and September, a slight dominance in the number of SG was recorded, with no differences measured among the cells in different developmental stages. The males studied exhibited a low gonadosomatic index (0.92% ± 0.27%). A significant reduction was recorded in motility (MOT), progressive motility (pMOT), and in most of the kinetic parameters (distance curved line-DCL, curvilinear velocity-VCL, straight line velocity-VSL, and beat cross frequency-BCF). The spermiation of males could have started in May and conceivably lasted until the end of June. It is recommended to increase sperm quality and quantity before cryopreservation.

Morphology of the male reproductive system and sperm of Leptoglossus zonatus (Dallas, 1852) (Heteroptera: Coreidae).

Taxonomic data on Coreidae have been fragmented over time and need to be revised. Likewise, data related to the development of germ cells and the features of the male reproductive system, including sperm, will contribute to understanding the biological mechanisms of reproduction and the systematics of its representatives. Aiming to provide these data, we describe the morphology of the male reproductive system and spermatozoa of Leptoglossus zonatus using light and transmission electron microscopies, respectively. Each of the two testes is surrounded by a bright red-pigmented sheath and formed by seven follicles arranged side by side. The two vasa deferentia are filled with individualized sperm, especially in their final portion, which is dilated and curved. After dilation, the vasa deferentia receive the ducts of the accessory glands of mesodermal origin. The other unpaired accessory gland is of ectodermal origin and opens into the ejaculatory duct. Both glandular types are densely coiled and have lumens filled with secreted material. Testicular follicles contain cysts with germ cells at different stages of spermatogenesis, indicating continuous production of gametes throughout adult life. Mature sperm measure around 310 μm long, with a nucleus of 36 μm and a flagellum formed only by an axoneme of 9 + 9 + 2 microtubules and two symmetrical mitochondrial derivatives. Like the sperm of other Heteroptera, the acrosome has a single structure (without perforatorium), there are no accessory bodies in the flagella, and the mitochondrial derivatives are connected to the axonemes, supporting the synapomorphic condition of these characteristics for this suborder of bedbugs. RESEARCH HIGHLIGHTS: The Leptoglossus zonatus sperm are slender and long, about 310 μm in length, and a nucleus 36 μm long. Spermatogenesis occurs throughout adult life and equally in the seven testicular follicles. The centriole adjunct in L. zonatus sperm does not give rise to accessory bodies. The ectodermal gland produces a filamentous secretion, whereas in the ectodermal sac, the secretion is globular.

Effect of Temperature on the Development of Stages of Spermatogenesis and the Functionality of Sertoli Cells In Vitro.

Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal spermatogenesis occurs in vivo at 34 °C to 35 °C, and high temperatures are known to cause male infertility. The aim of the present study was to examine the effect of temperature (35 °C compared to 37 °C) on the viability/apoptosis of developed cells, on the development of different stages of spermatogenesis in 3D in vitro culture conditions, and the functionality of Sertoli cells under these conditions. We used isolated cells from seminiferous tubules of sexually immature mice. The cells were cultured in methylcellulose (as a three-dimensional (3D) in vitro culture system) and incubated in a CO2 incubator at 35 °C or 37 °C. After two to six weeks, the developed cells and organoids were collected and examined for cell viability and apoptosis markers. The development of different stages of spermatogenesis was evaluated by immunofluorescence staining or qPCR analysis using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate the functionality of Sertoli cells were assessed by qPCR analysis. The developed organoids were examined by a confocal microscope. Our results show that the percentages and/or the expression levels of the developed pre-meiotic, meiotic, and post-meiotic cells were significantly higher at 35 °C compared to those at 37 °C, including the expression levels of the androgen receptor, the FSH receptor, transferrin, the androgen-binding protein (ABP), and the glial-derived nerve growth factor (GDNF) which were similarly significantly higher at 35 °C than at 37 °C. The percentages of apoptotic cells (according to acridine orange staining) and the expression levels of BAX, FAS, and CASPAS 3 were significantly higher in cultures incubated at 37 °C compared to those incubated at 35 °C. These findings support the in vivo results regarding the negative effect of high temperatures on the process of spermatogenesis and suggest a possible effect of high temperatures on the viability/apoptosis of spermatogenic cells. In addition, increasing the temperature in vitro also impaired the functionality of Sertoli cells. These findings may deepen our understanding of the mechanisms behind optimal conditions for normal spermatogenesis in vivo and in vitro.

Ultrastructural study: in vitro and in vivo differentiation of mice spermatogonial stem cells.

Mouse testicular tissue is composed of seminiferous tubules and interstitial tissue. Mammalian spermatogenesis is divided into three stages: spermatocytogenesis (mitotic divisions) in which spermatogonial stem cells (SSCs) turn into spermatocytes, followed by two consecutive meiotic divisions in which spermatocytes form spermatids. Spermatids differentiate into spermatozoa during spermiogenesis. Various factors affect the process of spermatogenesis and the organization of cells in the testis. Any disorder in different stages of spermatogenesis will have negative effects on male fertility. The aim of the current study was to compare the in vitro and in vivo spermatogenesis processes before and after transplantation to azoospermic mice using ultrastructural techniques. In this study, mice were irradiated with single doses of 14 Gy 60Co radiation. SSCs isolated from neonatal mice were cultured in vitro for 1 week and were injected into the seminiferous tubule recipient's mice. Testicular cells of neonatal mice were cultured in the four groups on extracellular matrix-based 3D printing scaffolds. The transplanted testes (8 weeks after transplantation) and cultured testicular cells in vitro (after 3 weeks) were then processed for transmission electron microscopy studies. Our study's findings revealed that the morphology and ultrastructure of testicular cells after transplantation and in vitro culture are similar to those of in vivo spermatogenesis, indicating that spermatogenic cell nature is unaltered in vitro.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced maturation of spermatogonial cells from prepubertal mice in vitro is enhanced by testosterone.

Spermatogenesis is the complicated process of sperm generation. During this process, spermatogonial cells proliferate and differentiate via meiotic and post-meiotic stages to produce mature sperm. This process is under the regulation of testicular autocrine/paracrine factors. In addition, endocrine factors are crucial to complete spermatogenesis. We aimed to localize granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFR) in testicular cells and further evaluate its involvement in the development of spermatogenesis in vitro. Weisolated cells from seminiferous tubule cells of seven-day-old mice and cultured them in vitro using a methylcellulose culture system (MCS), in the presence of GM-CSF and/or testosterone for four weeks. The cells were then examined for markers of different stages of spermatogenesis by immunofluorescence staining and/or qPCR analyses. Our results revealed the presence of GM-CSF and GM-CSFR in testicular cells (premeiotic and meiotic cells as well as somatic cells; Leydig and Sertoli cells). We further demonstrated the development of colonies/spheroids in the MCS which contained pre-meiotic, meiotic, and post-meiotic cells. The addition of GM-CSF to the MCS significantly increased the percentage of pre-meiotic and meiotic cells compared to control. Furthermore, the addition of GM-CSF and testosterone together significantly increased the percentage of cells in the post-meiotic stage compared to the addition of each separately. In conclusion, our results indicate that testicular cells express GM-CSF/GM-CSFR, and that GM-CSF is involved in the development of different stages of spermatogenesis in vitro. Furthermore, testosterone enhances the development of spermatogenic cells and potentiates the effect of GMCSF on the development of post-meiotic cells. These findings provide evidence that GM-CSF and testosterone are involved in the development of spermatogenesis in vitro and in vivo. In brief: Testicular somatic and germ cells express GM-CSF and GM-CSFR. Our study suggests that testicular GM-CSF is involved in the development of spermatogenesis, which is potentiated by testosterone.

P-420 Involvement of Interluekin-6 (IL-6) in the development of different stages of spermatogenesis in vitro, using spermatogonial cells isolated from normal and busulfan-treated immature mice.

Abstract Study question Can Interluekin-6 (IL-6) induce spermatogonial cells from seminiferous tubules of normal or busulfan-treated mice to develop spermatogenesis in vitro using methylcellulose culture system (MCS)? Summary answer IL-6 induced the development of spermatogonial cells from seminiferous tubules of normal or busulfan-treated mice to premeiotic, meiotic and post-meiotic cells in vitro. What is known already Spermatogenesis is a complicated process of sperm generation. During this process spermatogonial cells proliferate and differentiate to meiotic, postmeiotic stages that continue in spermiogenesis to generate mature sperm. This process is under regulation of autocrine and paracrine factors provided by developed germ cells and somatic cells such as Sertoli, peritubular and Leydig cells. These microenvironmental factors are modified following pathological condition, such as chemotherapy, which may lead to subfertility or sterility. Different in vitro culture systems were used to induce the development of complete spermatogenesis in vitro; however, this was not yet achieved. Study design, size, duration Sexually immature mice (7-day-old) were used as normal or were intraperitoneally (i.p) injected with busulfan (45 mg/kg) to isolate cells from their seminiferous tubules (STs). Cells were enzymatically isolated from the STs of the mice and were cultured in methylcellulose culture system (MCS), as a 3-dimensinal in vitro system. Fresh media without (CT) or with IL-6 were added to the cultures from the beginning, and after two weeks. The cultures were determined after 4 weeks Participants/materials, setting, methods Isolated cells from the STs were cultured (2x105/well/0.5ml) in MCS that contained StemPro-34 medium, KSR, rEGF, rGDNF, rLIF, and r-bFGF and in the presence/absence of IL-6 (1,10 or 100 pg/ml) and incubated for four weeks in a CO2 incubator at 37°C. The developed cells and colonies/organoids were examined microscopically and quantified for the development of cells of the different stages of spermatogenesis and Sertoli cells functional markers by immunofluorescence staining (IF) and/or qPCR analyses. Main results and the role of chance Addition of IL-6 to MCS that contained isolated cells from STs of normal immature mice significantly increased the development of premeiotic cells (VASA), meiotic (BOULE) and meiotic/postmeiotic cells (ACROSIN) as examined by specific IF and/or qPCR analyses. Addition of IL-6 also differently affected the expression levels of Sertoli cell functionality markers (androgen receptor, androgen binding protein, transferrin, GDNF, FSHR). Furthermore, addition of IL-6 to isolated cells from STs of busulfan-treated immature mice also increased the development of VASA-, BOULE- and ACROSIN-positive cells (as examined by IF and qPCR analyses). However, the effect of IL-6 was more potent in the development of different stages of spermatogenesis in in vitro cultures that contained isolated cells from STs of normal compared busulfan-treated mice. Also, addition of IL-6 distinctly affected the expression levels of Sertoli cell functional markers in cultures of normal and busulfan-treated mice. Limitations, reasons for caution This in-vitro culture used animal models. The bio-activity of the developed post-meiotic needs to be confirmed Wider implications of the findings These findings indicate the possible involvement of IL-6 in the development of spermatogenesis in vitro. This effect could be directly on spermatogonial cells and/or through Sertoli cells. Our result could suggest IL-6 as a potential factor to be used in developing future in vitro therapeutic strategies for male fertility preservation Trial registration number not applicable

In vitro complete differentiation of human spermatogonial stem cells to morphologic spermatozoa using a hybrid hydrogel of agarose and laminin

Spermatogenesis refers to the differentiation of the spermatogonial stem cells (SSCs) located in the base seminiferous tubules into haploid spermatozoa. Prerequisites for in vitro spermatogenesis include an extracellular matrix (ECM), paracrine factors, and testicular somatic cells which play a supporting role for SSCs. Thus, the present study evaluated the potential of co-culturing Sertoli cells and SSCs embedded in a hybrid hydrogel of agarose and laminin, the main components of the ECM. Following the three–week conventional culture of human testicular cells, the cells were cultured in agarose hydrogel or agarose/laminin one (hybrid) for 74 days. Then, immunocytochemistry, real-time PCR, electron microscopy, and morphological staining methods were applied to analyze the presence of SSCs, as well as the other cells of the different stages of spermatogenesis. Based on the results, the colonies with positive spermatogenesis markers were observed in both culture systems. The existence of the cells of all three phases of spermatogenesis (spermatogonia, meiosis, and spermiogenesis) was confirmed in the two groups, while morphological spermatozoa were detected only in the hybrid hydrogel group. Finally, a biologically improved 3D matrix can support all the physiological activities of SSCs such as survival, proliferation, and differentiation.

Single-cell transcriptome sequencing reveals Wolbachia-mediated modification in early stages of Drosophila spermatogenesis.

Wolbachia are the most widely distributed intracellular bacteria, and their most common effect on host phenotype is cytoplasmic incompatibility (CI). A variety of models have been proposed to decipher the molecular mechanism of CI, among which the host modification (HM) model predicts that Wolbachia effectors play an important role in sperm modification. However, owing to the complexity of spermatogenesis and testicular cell-type heterogeneity, whether Wolbachia have different effects on cells at different stages of spermatogenesis or whether these effects are linked with CI remains unknown. Therefore, we used single-cell RNA sequencing to analyse gene expression profiles in adult male Drosophila testes that were infected or uninfected by Wolbachia. We found that Wolbachia significantly affected the proportion of different types of germ cells and affected multiple metabolic pathways in germ cells. Most importantly, Wolbachia had the greatest impact on germline stem cells, resulting in dysregulated expression of genes related to DNA compaction, and Wolbachia infection also influenced the histone-to-protamine transition in the late stage of sperm development. These results support the HM model and suggest that future studies on Wolbachia-induced CI should focus on cells in the early stages of spermatogenesis.

Gap junctions mediate discrete regulatory steps during fly spermatogenesis.

Gametogenesis requires coordinated signaling between germ cells and somatic cells. We previously showed that Gap junction (GJ)-mediated soma-germline communication is essential for fly spermatogenesis. Specifically, the GJ protein Innexin4/Zero population growth (Zpg) is necessary for somatic and germline stem cell maintenance and differentiation. It remains unknown how GJ-mediated signals regulate spermatogenesis or whether the function of these signals is restricted to the earliest stages of spermatogenesis. Here we carried out comprehensive structure/function analysis of Zpg using insights obtained from the protein structure of innexins to design mutations aimed at selectively perturbing different regulatory regions as well as the channel pore of Zpg. We identify the roles of various regulatory sites in Zpg in the assembly and maintenance of GJs at the plasma membrane. Moreover, mutations designed to selectively disrupt, based on size and charge, the passage of cargos through the Zpg channel pore, blocked different stages of spermatogenesis. Mutations were identified that progressed through early germline and soma development, but exhibited defects in entry to meiosis or sperm individualisation, resulting in reduced fertility or sterility. Our work shows that specific signals that pass through GJs regulate the transition between different stages of gametogenesis.

Seasonal changes in the expression of molecular markers of stallion germ cells

The economic impacts of infertility and subfertility of stallions greatly influence the horse breeding industry. Self-renewal and differentiation of spermatogonial stem cells are the initial processes to maintain an adequate sperm population. Thus, understanding these processes may provide useful information to reveal the causes and remedies of subfertile and infertile stallions. Stallions are seasonal breeders. About 50% of the sperm population is reduced during the non-breeding season (NBS) in stallions. The seasonal regulation of spermatogenesis renders stallions as ideal models to understand the process of sperm production. Furthermore, comparing internal and external factors related to spermatogenesis during the breeding season (BS) and NBS may provide a solution for subfertile/infertile stallions. It is especially pertinent to study the expression pattern of different protein markers during undifferentiated, differentiating, and differentiated spermatogonia. Deleted in azoospermia-like (DAZL), undifferentiated cell transcription factor 1 (UTF-1), and protein gene product 9.5 (PGP9.5) are the molecular markers expressed at different stages of spermatogenesis. However, whether the expression pattern of these molecular markers is similar throughout the year in stallion remains undetermined. The objectives of this study were to (1) investigate the expression pattern and localization of DAZL, UTF-1, and PGP9.5 within seminiferous tubules and (2) evaluate the relative mRNA levels of these three germ cell markers in stallion testes during BS and NBS. Immunohistochemistry was performed to check and compare the expression pattern and localization of DAZL, UTF-1, and PGP9.5 antibodies. Reverse transcription-quantitative PCR analysis was performed to calculate the relative mRNA expression levels in the testes. Testicular tissues from thoroughbred stallions were collected during routine castration that was carried out in field conditions. Immunostaining of germ cells with DAZL and UTF-1 in BS and NBS were not significantly different. However, the relative mRNA expression levels of DAZL and UTF-1 were significantly different in both groups. Interestingly, the immunolabeling and the relative mRNA expression of PGP9.5 were significantly different between BS and NBS. From these results, it is hypothesized that the expression level of these putative molecular markers might be gonadotropin-dependent in stallion testes.

A systematic review of retinoic acid in the journey of spermatogonium to spermatozoa: From basic to clinical application

Background: Retinoic acid plays an essential role in testicular development and functions, especially spermatogenesis. We have reviewed the role of retinoic acid from basic (molecular) to clinical application. Methods: A search was conducted in the online database including PubMed, Google Scholar, and Scopus for English studies published in the last eight years about this issue. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines in assessing the studies we are going to investigate. Results: Studies indicated that retinoic acid plays an essential role during pluripotent stem cell migration and lineage commitment, cell differentiation, apoptosis, stem cell number regulation, and maturation arrest in spermatogenic cells. Retinoic acid can also affect related protein expression and signaling pathways at different stages of spermatogenesis. Four studies have applied retinoic acid to humans, all of them in the single-arm observational study. The results look promising but need further research with more controlled study methods, randomization, and large samples. Conclusions: This current systematic review emphasizes a novel retinoic acid mechanism that has not been well described in the literature previously on its functions during the first seven days of spermatogenesis, leading to new directions or explanations of male infertility cause and treatments as a part of reproductive health care.

P-081 Effect of chemotherapy (cytarabine) and acute myeloid leukemia on the development of spermatogenesis, spermatogonial microenvironment and inflammatory factors at the adult age of immature-treated mice

Abstract Study question How do AML and cytarabine affect the development of spermatogenesis, germ cell microenvironment and testicular inflammatory factors at the adult age of immature-treated mice? Summary answer AML and cytarabine significantly damaged the normal histology of the seminiferous tubules and impaired different stages of spermatogenesis, testicular growth/inflammatory factor in the adult age. What is known already The effect of different types of cancers on male fertility was reported. AML constitutes around 20% of the diagnosed childhood leukemia. Cytarabine is used as anti-AML treatment. Cytarabine is involved in reduction of testes growth and induces testicular atrophy in adults. In adult AML patients, it was shown that AML affects semen parameters even before chemotherapy treatment. Furthermore, a significant reduction in semen quality of thawed cryopreserved semen from leukemia patients compared to control patients was reported. However, the effect of AML and cytarabine before puberty on the development of spermatogenesis in adult age was not yet reported. Study design, size, duration Two-week-old males C57/BLACK mice were used. 1. Control group – injected with saline. 2. AML group – injected (intraperitoneal; i.p) with 3x104 AML cells/100 ml (murine C-1498 AML cell line). 3. Cytarabine group - three injections of cytarabine (140 mg/kg/100 ml) were performed every 12 hours. 4. AML+cytarabine group – cytarabine was i.p injected 24 hours after AML injection (injections were performed every 12 hours 3 times). Mice were sacrificed 1,2,3,4,5 weeks after AML injection. Participants/materials, setting, methods Testes were removed, weighted and fixed in Bouin’s solution for histological evaluation, or kept at -70°C for RNA extraction. The presence of premeiotic (SALL4, PLZF), meiotic (CREM-1) and postmeiotic (ACROSIN) cells were examined by immunofluorescence staining analysis and/or RNA expression by qPCR analysis. Main results and the role of chance We have successfully developed a system of AML in immature mice by i.p injection of C1498 cells. Cytarabine prolonged life of AML-treated mice from 2.5 weeks to 4 weeks. AML had no effect on both body and testicular weight. However, cytarabine significantly reduced testicular weight (around 50% of the control in the first 3 weeks after treatment, and around 30% of the control from 3-4.5 weeks after treatment; p < 0.001). One and/or 2 weeks after the treatment showed that both AML and cytarabine alone or in combination significantly decreased seminiferous tubules normal histology compared to control, (30%, 20%, 10% respectively) (p < 0.001). Cytarabine, but not AML significantly increased the expression of PLZF and SALL4 after 2 weeks compared to control (p < 0.001). However, each treatment significantly increased the percentages of seminiferous tubules with apoptotic cells compared to control (p < 0.05). AML or cytarabine significantly decreased the percentages of tubules with meiotic and postmeiotic markers compared to control (p < 0.05). Also, AML or cytarabine significantly decreased the testicular protein levels of GDNF, CSF-1 (p < 0.01) but did not affect LIF compared to control. Furthermore, AML or cytarabine significantly decreased the protein levels of IL-10. However, AML significantly increased IL-6 when cytarabine increased it compared to control (p < 0.01). Limitations, reasons for caution Animal model of leukemia may behave different from human model. Wider implications of the findings We successfully developed AML disease in immature mice, and demonstrated its impairment the normal histology of the seminiferous tubules and spermatogenesis in the adult age. We showed that AML and cytarabine affect testicular microenvironmental and inflammatory factors. Our results may assist in development future therapeutic strategies for male fertility preservation. Trial registration number NON

O-264 Testosterone potentiates the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the maturation of spermatogonial cells in vitro from prepubertal mice

Abstract Study question Can granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone induce spermatogonial cells to develop spermatogenesis in vitro using methylcellulose culture system (MCS)? Summary answer GM-CSF and testosterone induced the development of premeiotic and meiotic cells in vitro. Testosterone also induced and potentiate the development of post-meiotic cells by GM-CSF. What is known already Spermatogenesis is a complicated process of sperm generation. During this process spermatogonial cells proliferate and differentiate to meiotic, postmeiotic stages that continue in spermiogenesis to generate mature sperm. This process is under regulation of autocrine and paracrine factors provided by developed germ cells and somatic cells such as Sertoli, peritubular and Leydig cells. In addition, endocrine factors such as FSH, LH and testosterone are crucial for complete spermatogenesis. Different in vitro culture systems were used to induce the development of complete spermatogenesis in vitro, however this was not yet achieved. Study design, size, duration Sexually immature mice (7-day-old) were used to isolated cells from the seminiferous tubules. These isolated ells were cultured in methylcellulose culture system (MCS) as a 3-dimensinal in vitro system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone were added in the beginning of the cultures and after two weeks. The cultures were determined after 4 weeks. Participants/materials, setting, methods Cells were enzymatically isolated from the seminiferous tubules and cultured (2x105/well/0.5ml) in MCS that contained StemPro-34 medium, KSR, rEGF, rGDNF, rLIF, and r-bFGF. The cultures were grown in the presence/absence of GM-CSF (0.1 or 1ng/ml) and/or testosterone (10-7M) and incubated for four weeks in a CO2 incubator at 37°C. The developed cells and colonies/spheroids were examined microscopically, and quantified the developing cells of the different stages of spermatogenesis by immunofluorescence staining (IF) and/or qPCR analyses Main results and the role of chance Our results show the presence of GM-CSF and its receptor (GM-CSFR) in different testicular cells. We demonstrate the development of colonies/spheroids which contain premeiotic, meiotic and post-meiotic cells in MCS, as examined by IF and qPCR analyses. The percentage of pre-meiotic and miotic stage in the cultures with GM-CSF were significantly higher than those in the control group (p < 0.001), and the percentage of cells of the post-meiotic stage were significantly higher in the cultures with GM-CSF and testosterone together compared to each one separately (p < 0.001). In conclusion, testicular cells express GM-CSF/GM-CSFR, and GM-CSF affects only the development of premeiotic and meiotic cells in vitro. In addition, testosterone not only increased the development of cells from the premeiotic, meiotic and post-meiotic stages, but it also potentiated the effect of GM-CSF on the development of post-meiotic cells in vitro. Limitations, reasons for caution no limitation Wider implications of the findings These findings providing evidence that GM-CSF and testosterone could be used to improve the maturation of spermatogonial cells in vitro and that they might play a role in the maturation process of spermatogonial cells in vivo. Trial registration number not applicable

Spatiotemporal Regulation of a Single Adaptively Evolving Trans-Regulatory Element Contributes to Spermatogenetic Expression Divergence in Drosophila.

Due to extensive pleiotropy, trans-acting elements are often thought to be evolutionarily constrained. While the impact of trans-acting elements on gene expression evolution has been extensively studied, relatively little is understood about the contribution of a single trans regulator to interspecific expression and phenotypic divergence. Here, we disentangle the effects of genomic context and miR-983, an adaptively evolving young microRNA, on expression divergence between Drosophila melanogaster and D. simulans. We show miR-983 effects promote interspecific expression divergence in testis despite its antagonism with the often-predominant context effects. Single-cyst RNA-seq reveals that distinct sets of genes gain and lose miR-983 influence under disruptive or diversifying selection at different stages of spermatogenesis, potentially helping minimize antagonistic pleiotropy. At the round spermatid stage, the effects of miR-983 are weak and distributed, coincident with the transcriptome undergoing drastic expression changes. Knocking out miR-983 causes reduced sperm length with increased within-individual variation in D. melanogaster but not in D. simulans, and the D. melanogaster knockout also exhibits compromised sperm defense ability. Our results provide empirical evidence for the resolution of antagonistic pleiotropy and also have broad implications for the function and evolution of new trans regulators.

Effect of Palm Pollen and Pomegranate Peel Supplementation on Sperm Development in Male Rabbits.

The use of plants, plant extracts, and folk remedies for the treatment of human diseases has been known as traditional medicine. It is approved that the use of herbal medicine dates far back to 60,000 years ago in Iraq. This study aimed to investigate the possible effects of pomegranate and palm pollen, as two powerful herbal remedies with antioxidant, anti-inflammatory, and anti-bacterial effects, on fertility in the male rabbits. A total of 15 healthy New Zealand White male adult rabbits were randomly assigned to three groups (n=5 in each group). Group A: 5 rabbits did not receive herbal treatment (control group). Group B: 5 rabbits were treated with palm pollen at a dose of 2 g by oral gavage daily. Group C: 5 rabbits were treated with pomegranate peel (2 g) treated by oral gavage daily. All animals were euthanized 90 days after the beginning of the experiment. Following the euthanasia, the testis was removed. For sperm recovery, the epididymis was dissected from the testicles and the tail of the epididymis was minced in the sperm washing medium and incubated for 15 min in an atmosphere of humidified 5% CO2, 95% The samples were evaluated by using computerized semen analyzer apparatus (CASA). After 90 days, the treated groups with palm pollen and pomegranate peel (groups B and C) demonstrated a significant increase in the sperm concentration, compared to group (A). Moreover, the recorded data revealed that the sperm concentration in group C was significantly higher than that in group B. The study indicated that the total sperm motility was increased significantly in groups B and C, as compared to that in the control group, while the total sperm motility in group C increased significantly, compared to group B. The histological evolutions signified that in the control group, the epithelial of seminiferous tubules were at different stages of spermatogenesis, the spermatocytogenesis (primary and secondary) with the spermogenesis (spermatids) were found in a cluster at the cavity of the tubule. Furthermore, the results of histological evaluations in groups B and C were distinguished by a significant augmentation of the seminiferous tubules with primary and secondary spermatocytes, as well as some degrees of spermogenesis (round spermatids), compared to group A. Moreover, increased angiogenesis was observed in groups B and C. In conclusion, the results pointed out that herbal remedies have a significant effects in the treatment of reproductive disorders.

Immunolocalization of Vasa, PIWI, and TDRKH proteins in male germ cells during spermatogenesis of the teleost fish Poecilia reticulata

Vasa, PIWI and TDRKH are conserved components of germ granules that in metazoans are involved in germline specification and differentiation, as documented by mutational experiments in some model animals. So far, investigations on PIWI during spermatogenesis of fish has been limited to a few species, and no information is available for TDRKH, another protein involved in the piRNA pathway. In this study, the immunolocalization of these three germline determinants was analyzed in male gonads of the teleost fish Poecilia reticulata to document their localization pattern in the different stages of germ cell differentiation.To analyze their distribution pattern during the different stages of spermatogenesis we performed immunohistochemistry (IHC) and immunofluorescence (IF) assays using primary polyclonal antibodies after testing their specificity with Western Blot. Moreover, sections of testis stained with haematoxylin and eosin clarified the structural organization of P. reticulata testis, while the use of the confocal microscope and the nuclear staining clarified the different stages of germ cell differentiation during spermatogenesis.The results showed that Vasa, PIWI and TDRKH were specifically immunolocalized in the germ cells of P. reticulata, with no specific signal detected in Sertoli cells and in other somatic cells of the gonad. These markers were detected in all stages of differentiation from early spermatogonia to advanced spermatids. Vasa staining was the strongest in spermatogonia, and then decreases throughout differentiation. Instead, both PIWI and TDRKH staining increases during differentiation, and their distribution pattern, similar to what observed in the mouse, suggests their concerted participation in the piRNA pathway also in this fish.

PAZ domain is critical for spermatogenesis in chicken

Piwi-like protein 1 (PIWIL1) plays a crucial role in stem cell proliferation, embryogenesis, growth, and development. We aimed to unravel the function of PIWIL1 and its Piwi/Argonaute/Zwille (PAZ) domain in chicken embryogenesis. The expression of PIWI1 at different stages of spermatogenesis was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the PAZ domain was mutated based on its 3D structure model using the clustered regularly interspaced short palindromic repeats Cas9 (CRISPR/Cas9) technology. The results indicated that PIWIL1 mRNA was specifically expressed in spermatogonium cells undergoing meiosis. After targeting the PAZ domain (300–370 amino acid residues), we obtained two mutant DF-1 cell clones with 23-bp and 8-bp deletions. Injection of the pCMV-Cas9-puro-sgRNA-2 construct into 2.5-day embryos resulted in generation of 19 different PAZ mutants (13 males and 6 females), which showed delayed hatching, reduced quality of semen, and decreased expression of PIWIL1 and SOX2 at embryonic days 5 and 18. However, we could not obtain PAZ double knockout (KO) chickens by crossing of the F0 generation, suggesting that PAZ double KO may halt embryonic development. Our results indicate that PIWIL1 plays an important role in meiosis and that PAZ mutations can lead to decreased sperm quality, whereas its double KO may arrest embryogenesis in chicken.

RDC complex executes a dynamic piRNA program during Drosophila spermatogenesis to safeguard male fertility.

piRNAs are small non-coding RNAs that guide the silencing of transposons and other targets in animal gonads. In Drosophila female germline, many piRNA source loci dubbed "piRNA clusters" lack hallmarks of active genes and exploit an alternative path for transcription, which relies on the Rhino-Deadlock-Cutoff (RDC) complex. RDC was thought to be absent in testis, so it remains to date unknown how piRNA cluster transcription is regulated in the male germline. We found that components of RDC complex are expressed in male germ cells during early spermatogenesis, from germline stem cells (GSCs) to early spermatocytes. RDC is essential for expression of dual-strand piRNA clusters and transposon silencing in testis; however, it is dispensable for expression of Y-linked Suppressor of Stellate piRNAs and therefore Stellate silencing. Despite intact Stellate repression, males lacking RDC exhibited compromised fertility accompanied by germline DNA damage and GSC loss. Thus, piRNA-guided repression is essential for normal spermatogenesis beyond Stellate silencing. While RDC associates with multiple piRNA clusters in GSCs and early spermatogonia, its localization changes in later stages as RDC concentrates on a single X-linked locus, AT-chX. Dynamic RDC localization is paralleled by changes in piRNA cluster expression, indicating that RDC executes a fluid piRNA program during different stages of spermatogenesis. These results disprove the common belief that RDC is dispensable for piRNA biogenesis in testis and uncover the unexpected, sexually dimorphic and dynamic behavior of a core piRNA pathway machinery.

Polyubiquitin gene Ubb is required for upregulation of Piwi protein level during mouse testis development

Testis development, including early embryonic gonad formation and late postnatal spermatogenesis, is essential for the reproduction of higher metazoans to generate fertile gametes, called sperm. We have previously reported that the polyubiquitin gene Ubb is required for fertility in both male and female mice. In particular, the Ubb-null male mice showed an azoospermia phenotype due to arrest of spermatogenesis at the pachytene stage. Here, we analyzed the whole testis proteome at postnatal day 20 to define the molecular mediators of the male-infertility phenotype caused by Ubb knockout. From the identified proteome, 564 proteins were significantly and differentially expressed in Ubb-knockout testes and, among these, 36 downregulated proteins were involved at different stages of spermatogenesis. We also found that levels of piRNA metabolic process-related proteins, including Piwil2 and Tdrd1, were downregulated in Ubb-null testes through functional gene ontology analysis. Further, protein–protein interaction mapping revealed that 24 testis development-related proteins, including Hsp90aa1, Eef1a1, and Pabpc1, were directly influenced by the depletion of ubiquitin. In addition, the reduced mRNA levels of these proteins were observed in Ubb-knockout testes, which closely resembled the global downregulation of piRNA-metabolic gene expression at the transcriptional and post-transcriptional levels. Together with proteomic and transcriptional analyses, our data suggest that Ubb expression is essential for the maintenance of testicular RNA-binding regulators and piRNA-metabolic proteins to complete spermatogenesis in mice.