SPSL1 is essential for spermatophore formation and sperm activation in Spodoptera frugiperda.

The reproductive process in various species has undergone evolutionary adaptations at both the physiological and molecular levels, playing a significant role in maintaining their populations. In lepidopteran insects, the spermatophore is a unique structure formed in the female reproductive system, in which sperm storage and activation take place. It is known that the formation of the spermatophore is regulated by seminal fluid proteins derived from males. However, studies investigating the genetic mechanisms behind spermatophore formation in lepidopterans have been limited. In this study, our focus was on SPSL1, a gene that encodes a trypsin-type seminal fluid protein in Spodoptera frugiperda, a pest species with global invasive tendencies. Our findings revealed that SPSL1 expression was predominantly observed in the male reproductive tracts, and the disruption of this gene resulted in male sterility. Surprisingly, fluorescence analysis indicated that the absence of SPSL1 did not affect spermatogenesis or sperm migration within the male reproductive system. However, when females mated with SPSL1-mutant males, several defects were observed. These included disruptions in spermatophore formation, sperm activation in the copulatory bursae, and sperm migration into the spermathecae. Additionally, mass spectrometry analysis highlighted reduced levels of energy-related metabolites, suggesting that SPSL1 plays an essential role in promoting hydrolysis reactions during copulation. Consequently, our study demonstrates that SPSL1 is crucial for male fertility due to its functions in spermatophore formation and sperm activation. This research provides valuable insights into the genetic factors underlying reproductive processes in lepidopteran insects and sheds light on potential strategies for controlling invasive pest populations.

Developmental origins of mammalian spermatogonial stem cells: New perspectives on epigenetic regulation and sex chromosome function.

Male and female germ cells undergo genome-wide reprogramming during their development, and execute sex-specific programs to complete meiosis and successfully generate healthy gametes. While sexually dimorphic germ cell development is fundamental, similarities and differences exist in the basic processes governing normal gametogenesis. At the simplest level, male gamete generation in mammals is centred on the activity of spermatogonial stem cells (SSCs), and an equivalent cell state is not present in females. Maintaining this unique SSC epigenetic state, while keeping to germ cell-intrinsic developmental programs, poses challenges for the correct completion of spermatogenesis. In this review, we highlight the origins of spermatogonia, comparing and contrasting them with female germline development to emphasize specific developmental processes that are required for their function as germline stem cells. We identify gaps in our current knowledge about human SSCs and further discuss the impact of the unique regulation of the sex chromosomes during spermatogenesis, and the roles of X-linked genes in SSCs.

Notch pathway is required for protection against heat stress in spermatogonial stem cells in medaka.

Gamete production is a fundamental process for reproduction; however, exposure to stress, such as increased environmental temperature, can decrease or even interrupt this process, affecting fertility. Thus, the survival of spermatogonial stem cells (SSCs) is crucial for the recovery of spermatogenesis upon stressful situations. Here, we show that the Notch pathway is implicated in such survival, by protecting the SSCs against thermal stress. First, we corroborated the impairment of spermatogenesis under heat stress in medaka, observing an arrest in metaphase I at 10 days of heat treatment, an increase in the number of spermatocytes, and downregulation of ndrg1b and sycp3. In addition, at 30 days of treatment, an interruption of spermatogenesis was observed with a strong loss of spermatocytes and spermatids. Then, the exposure of adult males to thermal stress condition induced apoptosis mainly in spermatogenic and supporting somatic cells, with the exception of the germinal region, where SSCs are located. Concomitantly, the Notch pathway-related genes were upregulated, including the ligands (dll4, jag1-2) and receptors (notch1a-3). Moreover, during thermal stress presenilin enhancer-2 (pen-2), the catalytic subunit of γ-secretase complex of the Notch pathway was restricted to the germinal region of the medaka testis, observed in somatic cells surrounding type A spermatogonia (SGa). The importance of Notch pathway was further supported by an ex vivo approach, in which the inhibition of this pathway activity induced a loss of SSCs. Overall, this study supports the importance of Notch pathways for the protection of SSCs under chronic thermal stress.

Exosome-derived Small RNAs in mouse Sertoli cells inhibit spermatogonial apoptosis.

Spermatogenesis is a highly complicated biological process that occurs in the epithelium of the seminiferous tubules. It is regulated by a complex network of endocrine and paracrine factors. Sertoli cells (SCs) play a key role in spermatogenesis due to their production of trophic, differentiation, and immune-modulating factors. However, many of the molecular pathways of SC action remain controversial and unclear. Recently, many studies have focused on exosomes as an important mechanism of intercellular communication. We found that the exosomes derived from mouse SCs inhibited the apoptosis of primary spermatogonia. A total of 1016 miRNAs in SCs and 556 miRNAs in exosomes were detected using miRNA high-throughput sequencing. A total of 294 miRNAs were differentially expressed between SCs and exosomes. Furthermore, 19 tsRNA families appeared in SCs, while 6 tsRNA families appeared in exosomes. A total of 57 and 1 miRNAs (RPM >4) and 14 and 1 tsRNAs were exclusively expressed in SCs and exosomes, respectively. MiR-10b is one of the top ten exosomes with a relatively large enrichment of miRNA. Overexpression of miR-10b downregulates the expression of the target KLF4 to reduce spermatogonial apoptosis in primary spermatogonia or the C18-4 cell line.

Bud31-mediated alternative splicing is required for spermatogonial stem cell self-renewal and differentiation.

Alternative splicing (AS) is tightly regulated during cell differentiation and development. AS events are prevalent in the testis, but the splicing regulation in spermatogenesis remains unclear. Here we report that the spliceosome component Bud31 plays a crucial role during spermatogenesis in mice. Germ cell-specific knockout of Bud31 led to loss of spermatogonia and to male infertility. We further demonstrate that Bud31 is required for both spermatogonial stem cell pool maintenance and the initiation of spermatogenesis. SMART-seq revealed that deletion of Bud31 in germ cells causes widespread exon-skipping and intron retention. Particularly, we identified Cdk2 as one of the direct splicing targets of Bud31, knockout of Bud31 resulted in retention of the first intron of Cdk2, which led to a decrease in Cdk2 expression. Our findings suggest that Bud31-mediated AS within spermatogonial stem cells regulates the self-renewal and differentiation of male germ cells in mammals.

Zinc transporter ZIP12 maintains zinc homeostasis and protects spermatogonia from oxidative stress during spermatogenesis.

BACKGROUND: Overwhelming evidences suggest oxidative stress is a major cause of sperm dysfunction and male infertility. Zinc is an important non-enzymatic antioxidant with a wide range of biological functions and plays a significant role in preserving male fertility. Notably, zinc trafficking through the cellular and intracellular membrane is mediated by specific families of zinc transporters, i.e., SLC39s/ZIPs and SLC30s/ZnTs. However, their expression and function were rarely evaluated in the male germ cells. The aim of this study is to determine and characterize the crucial zinc transporter responsible for the maintenance of spermatogenesis. METHODS: The expression patterns of all 14 ZIP members were characterized in the mouse testis. qRT-PCR, immunoblot and immunohistochemistry analyses evaluated the ZIP12 gene and protein expression levels. The role of ZIP12 expression was evaluated in suppressing the sperm quality induced by exposure to an oxidative stress in a spermatogonia C18-4 cell line. Zip12 RNAi transfection was performed to determine if its downregulation altered cell viability and apoptosis in this cell line. An obese mouse model fed a high-fat-diet was employed to determine if there is a correlation between changes in the ZIP12 expression level and sperm quality. RESULTS: The ZIP12 mRNA and protein expression levels were higher than those of other ZIP family members in both the mouse testis and other tissues. Importantly, the ZIP12 expression levels were very significantly higher in both mice and human spermatogonia and spermatozoa. Moreover, the testicular ZIP12 expression levels significantly decreased in obese mice, which was associated with reduced sperm zinc content, excessive sperm ROS generation, poor sperm quality and male subfertility. Similarly, exposure to an oxidative stress induced significant declines in the ZIP12 expression level in C18-4 cells. Knockdown of ZIP12 expression mediated by transfection of a ZIP12 siRNA reduced both the zinc content and viability whereas apoptotic activity increased in the C18-4 cell line. CONCLUSIONS: The testicular zinc transporter ZIP12 expression levels especially in spermatogonia and spermatozoa are higher than in other tissues. ZIP12 may play a key role in maintaining intracellular zinc content at levels that reduce the inhibitory effects of rises in oxidative stress on spermatogonia and spermatozoa viability during spermatogenesis which help counteract declines in male fertility.

Localization and regulatory function of Yin Yang 1 (YY1) in chicken testis.

In mammals, Yin Yang 1 (YY1), a pervasively expressed transcription factor related to many biological processes as an activator or inhibitor of the transcription of various genes, plays a critical role in the development of male gonads and spermatogenesis. Although the role of YY1 on the development of male gonads and spermatogenesis in mammals has been reported, its function on chicken testis are yet to be clarified. In this study, we used immunofluorescence analysis to investigate the location of YY1 in chicken testis. In embryo testis, YY1 was detected in spermatogonia and Sertoli cells, while in adult testis, YY1 was shown to be expressed in spermatogenic cells and Sertoli cells, but not in spermatozoa. Furthermore, we investigated the regulatory functions of YY1 in chicken testicular Sertoli cells by combining overexpression with RNA-sequencing. Overexpression of YY1 in Sertoli cells revealed a total of 2955 differentially expressed genes involved in various biological processes, such as male gonad development and seminiferous tubule development. Overexpression of YY1 also caused significant differences in the expression of the androgen receptor gene and the inhibin ßA gene, two major genes involved in the regulation of spermatogonia in Sertoli cells. These observations indicate that YY1 may regulate the development and function of the gonads by affecting the secretion of cytokines and hormones in Sertoli cells to mediate the production and differentiation of spermatogonia.

The Proliferation of Pre-Pubertal Porcine Spermatogonia in Stirred Suspension Bioreactors Is Partially Mediated by the Wnt/ß-Catenin Pathway.

Male survivors of childhood cancer are at risk of suffering from infertility in adulthood because of gonadotoxic chemotherapies. For adult men, sperm collection and preservation are routine procedures prior to treatment; however, this is not an option for pre-pubertal children. From young boys, a small biopsy may be taken before chemotherapy, and spermatogonia may be propagated in vitro for future transplantation to restore fertility. A robust system that allows for scalable expansion of spermatogonia within a controlled environment is therefore required. Stirred suspension culture has been applied to different types of stem cells but has so far not been explored for spermatogonia. Here, we report that pre-pubertal porcine spermatogonia proliferate more in bioreactor suspension culture, compared with static culture. Interestingly, oxygen tension provides an avenue to modulate spermatogonia status, with culture under 10% oxygen retaining a more undifferentiated state and reducing proliferation in comparison with the conventional approach of culturing under ambient oxygen levels. Spermatogonia grown in bioreactors upregulate the Wnt/ ß-catenin pathway, which, along with enhanced gas and nutrient exchange observed in bioreactor culture, may synergistically account for higher spermatogonia proliferation. Therefore, stirred suspension bioreactors provide novel platforms to culture spermatogonia in a scalable manner and with minimal handling.

Successful Spermatogonial Stem Cells Transplantation within Pleuronectiformes: First Breakthrough at inter-family Level in Marine Fish.

As a promising biotechnology, fish germ cell transplantation shows potentials in conservation germplasm resource, propagation of elite species, and generation of transgenic individuals. In this study, we successfully transplanted the Japanese flounder (P. olivaceus), summer flounder (P. dentatus), and turbot (S. maximus) spermatogonia into triploid Japanese flounder larvae, and achieved high transplantation efficiency of 100%, 75-95% and 33-50% by fluorescence tracking and molecular analysis, respectively. Eventually, donor-derived spermatozoa produced offspring by artificial insemination. We only found male and intersex chimeras in inter-family transplantations, while male and female chimeras in both intra-species and intra-genus transplantations. Moreover, the intersex chimeras could mature and produce turbot functional spermatozoa. We firstly realized inter-family transplantation in marine fish species. These results demonstrated successful spermatogonial stem cells transplantation within Pleuronectiformes, suggesting the germ cells migration, incorporation and maturation within order were conserved across a wide range of teleost species.

Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock.

PURPOSE: Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in cancer patients. Whereas in livestock, it is used for mining the factors associated with male fertility. The review discusses the present status of SSC biology, methodologies developed for in vitro culture, and challenges ahead in establishing SSC technology for the propagation of superior germplasm with special reference to livestock. METHOD: Published literatures from PubMed and Google Scholar on topics of SSCs isolation, purification, characterization, short and long-term culture of SSCs, stemness maintenance, epigenetic modifications of SSCs, growth factors, and SSC cryopreservation and transplantation were used for the study. RESULT: The fine-tuning of SSC isolation and culture conditions with special reference to feeder cells, growth factors, and additives need to be refined for livestock. An insight into the molecular mechanisms involved in maintaining stemness and proliferation of SSCs could facilitate the dissemination of superior germplasm through transplantation and transgenesis. The epigenetic influence on the composition and expression of the biomolecules during in vitro differentiation of cultured cells is essential for sustaining fertility. The development of surrogate males through gene-editing will be historic achievement for the foothold of the SSCs technology. CONCLUSION: Detailed studies on the species-specific factors regulating the stemness and differentiation of the SSCs are required for the development of a long-term culture system and in vitro spermatogenesis in livestock. Epigenetic changes in the SSCs during in vitro culture have to be elucidated for the successful application of SSCs for improving the productivity of the animals.

Granulocyte Colony-Stimulating Factor Restored Impaired Spermatogenesis and Fertility in an AML-Chemotherapy Mice Model.

Leukemia and treatment of male patients with anticancer therapy (aggressive chemotherapy and/or radiotherapy) may lead to infertility or even permanent male sterility. Their mechanisms of spermatogenesis impairment and the decrease in male fertility are not yet clear. We showed that under acute myeloid leukemia (AML) conditions, alone and in combination with cytarabine (CYT), there was significant damage in the histology of seminiferous tubules, a significant increase in apoptotic cells of the seminiferous tubules, and a reduction in spermatogonial cells (SALL and PLZF) and in meiotic (CREM) and post-meiotic (ACROSIN) cells. In addition, we showed a significant impairment in sperm parameters and fertilization rates and offspring compared to control. Our results showed a significant decrease in the expression of glial cell line-derived neurotrophic factor (GDNF), macrophage colony-stimulating factor (MCSF) and stem cell factor (SCF) under AML conditions, but not under cytarabine treatment compared to control. In addition, our results showed a significant increase in the pro-inflammatory cytokine interleukin-1 (IL-1) alpha in whole testis homogenates in all treatment groups compared to the control. Increase in IL-1 beta level was shown under AML conditions. We identified for the first time the expression of GCSF receptor (GCSFR) in sperm cells. We showed that GCSF injection in combination with AML and cytarabine (AML + CYT + GCSF) extended the survival of mice for a week (from 6.5 weeks to 7.5 weeks) compared to (AML + CYT). Injection of GCSF to all treated groups (post hoc), showed a significant impact on mice testis weight, improved testis histology, decreased apoptosis and increased expression of pre-meiotic, meiotic and post- meiotic markers, improved sperm parameters, fertility capacity and number of offspring compared to the controls (without GCSF). GCSF significantly improved the spermatogonial niche expressed by increased the expression levels of testicular GDNF, SCF and MCSF growth factors in AML-treated mice and (AML + CYT)-treated mice compared to those groups without GCSF. Furthermore, GCSF decreased the expression levels of the pro-inflammatory cytokine IL-12, but increased the expression of IL-10 in the interstitial compartment compared to the relevant groups without GCSF. Our results show for the first time the capacity of post injection of GCSF into AML- and CYT-treated mice to improve the cellular and biomolecular mechanisms that lead to improve/restore spermatogenesis and male fertility. Thus, post injection of GCSF may assist in the development of future therapeutic strategies to preserve/restore male fertility in cancer patients, specifically in AML patients under chemotherapy treatments.

Mammalian in vitro gametogenesis.

Germ cells differentiate into sexually dimorphic gametes, oocytes, and spermatozoa, which unite to form new individuals. Accordingly, germ cell development entails intricate regulations of genome functions for genetic and epigenetic inheritance. The past decade has seen considerable advances in in vitro gametogenesis (IVG), which aims to recreate germ cell development from pluripotent stem cells (PSCs) in culture. Mouse PSCs can be induced into functional oocytes and spermatozoa, whereas human PSCs can be induced into early oocytes and prospermatogonia, promoting mechanistic understanding of mammalian germ cell development. The prospect for inducing human gametes with appropriate functions has been heightened, and such advances will create possibilities in reproductive medicine, including modeling infertility to explore remedies. The use of IVG-derived gametes for human reproduction will require careful legal and ethical discussions.

Germ Cell Derivation from Pluripotent Stem Cells for Understanding In Vitro Gametogenesis.

Assisted reproductive technologies (ARTs) have developed considerably in recent years; however, they cannot rectify germ cell aplasia, such as non-obstructive azoospermia (NOA) and oocyte maturation failure syndrome. In vitro gametogenesis is a promising technology to overcome infertility, particularly germ cell aplasia. Early germ cells, such as primordial germ cells, can be relatively easily derived from pluripotent stem cells (PSCs); however, further progression to post-meiotic germ cells usually requires a gonadal niche and signals from gonadal somatic cells. Here, we review the recent advances in in vitro male and female germ cell derivation from PSCs and discuss how this technique is used to understand the biological mechanism of gamete development and gain insight into its application in infertility.

The essential roles of Mps1 in spermatogenesis and fertility in mice.

Monopolar spindle 1 (MPS1), which plays a critical role in somatic mitosis, has also been revealed to be essential for meiosis I in oocytes. Spermatogenesis is an important process involving successive mitosis and meiosis, but the function of MPS1 in spermatogenesis remains unclear. Here, we generated Mps1 conditional knockout mice and found that Ddx4-cre-driven loss of Mps1 in male mice resulted in depletion of undifferentiated spermatogonial cells and subsequently of differentiated spermatogonia and spermatocytes. In addition, Stra8-cre-driven ablation of Mps1 in male mice led to germ cell loss and fertility reduction. Spermatocytes lacking Mps1 have blocked at the zygotene-to-pachytene transition in the prophase of meiosis I, which may be due to decreased H2B ubiquitination level mediated by MDM2. And the expression of many meiotic genes was decreased, while that of apoptotic genes was increased. Moreover, we also detected increased apoptosis in spermatocytes with Mps1 knockout, which may have been the reason why germ cells were lost. Taken together, our findings indicate that MPS1 is required for mitosis of gonocytes and spermatogonia, differentiation of undifferentiated spermatogonia, and progression of meiosis I in spermatocytes.

EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice.

The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

CG6015 controls spermatogonia transit-amplifying divisions by epidermal growth factor receptor signaling in Drosophila testes.

Spermatogonia transit-amplifying (TA) divisions are crucial for the differentiation of germline stem cell daughters. However, the underlying mechanism is largely unknown. In the present study, we demonstrated that CG6015 was essential for spermatogonia TA-divisions and elongated spermatozoon development in Drosophila melanogaster. Spermatogonia deficient in CG6015 inhibited germline differentiation leading to the accumulation of undifferentiated cell populations. Transcriptome profiling using RNA sequencing indicated that CG6015 was involved in spermatogenesis, spermatid differentiation, and metabolic processes. Gene Set Enrichment Analysis (GSEA) revealed the relationship between CG6015 and the epidermal growth factor receptor (EGFR) signaling pathway. Unexpectedly, we discovered that phosphorylated extracellular regulated kinase (dpERK) signals were activated in germline stem cell (GSC)-like cells after reduction of CG6015 in spermatogonia. Moreover, Downstream of raf1 (Dsor1), a key downstream target of EGFR, mimicked the phenotype of CG6015, and germline dpERK signals were activated in spermatogonia of Dsor1 RNAi testes. Together, these findings revealed a potential regulatory mechanism of CG6015 via EGFR signaling during spermatogonia TA-divisions in Drosophila testes.

Metabolic Requirements for Spermatogonial Stem Cell Establishment and Maintenance In Vivo and In Vitro.

The spermatogonial stem cell (SSC) is a unique adult stem cell that requires tight physiological regulation during development and adulthood. As the foundation of spermatogenesis, SSCs are a potential tool for the treatment of infertility. Understanding the factors that are necessary for lifelong maintenance of a SSC pool in vivo is essential for successful in vitro expansion and safe downstream clinical usage. This review focused on the current knowledge of prepubertal testicular development and germ cell metabolism in different species, and implications for translational medicine. The significance of metabolism for cell biology, stem cell integrity, and fate decisions is discussed in general and in the context of SSC in vivo maintenance, differentiation, and in vitro expansion.

Thy1-Positive Spermatogonia Suppress the Proliferation of Spermatogonial Stem Cells by Extracellular Vesicles In Vitro.

The self-renewal of mammalian spermatogonial stem cells (SSCs) supports spermatogenesis to produce spermatozoa, and this is precisely controlled in a stem niche microenvironment in the seminiferous tubules. Although studies have revealed the role of the surrounding factors in SSCs, little is known about whether the division of SSCs is controlled by extracellular vesicles. Here, extracellular vesicles were found in the basal compartment of seminiferous tubules in mouse, rat, rabbit and human testes. In the mice, the testicular extracellular vesicles are secreted by spermatogonia and are taken up by SSCs. Further, the extracellular vesicles from thy1-positive spermatogonia were purified by anti-Thy1-coupled magnetic beads, which suppress their proliferation of SSCs but do not lead to the apoptosis in vitro.

An mTORC1-dependent switch orchestrates the transition between mouse spermatogonial stem cells and clones of progenitor spermatogonia.

Spermatogonial stem cells (SSCs) sustain spermatogenesis by balancing self-renewal and initiation of differentiation to produce progenitor spermatogonia committed to forming sperm. To define the regulatory logic among SSCs and progenitors, we performed single-cell RNA velocity analyses and validated results in vivo. A predominant quiescent SSC population spawns a small subset of cell-cycle-activated SSCs via mitogen-activated protein kinase (MAPK)/AKT signaling. Activated SSCs form early progenitors and mTORC1 inhibition drives activated SSC accumulation consistent with blockade to progenitor formation. Mechanistically, mTORC1 inhibition suppresses transcription among spermatogonia and specifically alters expression of insulin growth factor (IGF) signaling in early progenitors. Tex14-/- testes lacking intercellular bridges do not accumulate activated SSCs following mTORC1 inhibition, indicating that steady-state mTORC1 signaling drives activated SSCs to produce progenitor clones. These results are consistent with a model of SSC self-renewal dependent on interconversion between activated and quiescent SSCs, and mTORC1-dependent initiation of differentiation from SSCs to progenitor clones.

The Expression Levels and Cellular Localization of Pigment Epithelium Derived Factor (PEDF) in Mouse Testis: Its Possible Involvement in the Differentiation of Spermatogonial Cells.

Pigment epithelium derived factor (PEDF) is a multifunctional secretory soluble glycoprotein that belongs to the serine protease inhibitor (serpin) family. It was reported to have neurotrophic, anti-angiogenic and anti-tumorigenic activity. Recently, PEDF was found in testicular peritubular cells and it was assumed to be involved in the avascular nature of seminiferous tubules. The aim of this study was to determine the cellular origin, expression levels and target cells of PEDF in testicular tissue of immature and adult mice under physiological conditions, and to explore its possible role in the process of spermatogenesis in vitro. Using immunofluorescence staining, we showed that PEDF was localized in spermatogenic cells at different stages of development as well as in the somatic cells of the testis. Its protein levels in testicular homogenates and Sertoli cells supernatant showed a significant decrease with age. PEDF receptor (PEDF-R) was localized within the seminiferous tubule cells and in the interstitial cells compartment. Its RNA expression levels showed an increase with age until 8 weeks followed by a decrease. RNA levels of PEDF-R showed the opposite trend of the protein. Addition of PEDF to cultures of isolated cells from the seminiferous tubules did not changed their proliferation rate, however, a significant increase was observed in number of meiotic/post meiotic cells at 1000 ng/mL of PEDF; indicating an in vitro differentiation effect. This study may suggest a role for PEDF in the process of spermatogenesis.