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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 CO 2 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.

Macrophage migration inhibitory factor (MIF) is a multifunctional molecule. MIF was originally identified as a T-cell-derived factor responsible for the inhibition of macrophage migration. In testicular tissue of adult rats, MIF is constitutively expressed by Leydig cells under physiological conditions. The aim of this study was to examine MIF levels in testicular homogenates from different aged mice, and the capacity of Sertoli cells to produce it. We also examined MIF involvement in spermatogonial cell migration. Similar levels of MIF protein were detected in testicular homogenates of mice of different ages (1-8-week-old). However, the RNA expression levels of MIF were high in 1-week-old mice and significantly decreased with age compared to 1-week-old mice. MIF was stained in Sertoli, Leydig cells, and developed germ cells in the seminiferous tubules. Isolated Sertoli cells from 1-week-old mice stained to MIF. Cultures of Sertoli cells from 1-week-old mice produced and expressed high levels of MIF which significantly decreased with age. MIF was localized in the cytoplasm and nucleus of Sertoli cell cultures isolated from 1-week-old mice; however, it was localized only in the cytoplasm and branches of cultures isolated from 8-week-old mice. MIFR was detected in GFRα1 and Sertoli cells. MIF could induce migration of spermatogonial cells, and this effect was synergistic with glial cell-line neurotrophic factor. Our results show, for the first time, the capacity of Sertoli cells to produce MIF under normal conditions and that MIFR expressed in GFRα1 and Sertoli cells. We also showed that MIF induced spermatogonial cell migration. J. Cell. Physiol. 232: 2869-2877, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The Interleukin-1 (IL-1) system has been suggested to be involved in the cell-cell cross talk within the testis. To investigate the testicular autocrine, paracrine and endocrine factors involved in the regulation of Sertoli cell functions, we have examined the capacity of Sertoli cell cultures, from immature mice, to produce IL-1alpha, IL-1beta and IL-1 receptor antagonist (IL-1ra) under in vitro cultures and in the presence of testicular physiological and pathological factors. Our investigation revealed that Sertoli cells produce large amounts of IL-1alpha, IL-1ra but not IL-1beta under basal culture conditions, as examined by ELISA and immunohistochemical staining. Liposaccharides (LPS), as well as IL-1alpha and IL-1beta were found to stimulate IL-1alpha and IL-1ra, but not IL-1beta production, in Sertoli cells from immature mice. Maximum concentration of IL-1alpha and of IL-1ra was observed after 2 and 8 h after the stimulation, respectively. The addition of IL-1ra to Sertoli cells did not alter their capacity to constitutively produce IL-1alpha. However, the stimulatory effects of recombinant IL-1alpha on IL-1alpha production by Sertoli cells were reversed by the concomitant addition of recombinant IL-1ra. FSH is capable to induce IL-1ra production in Sertoli cells in a dose-dependent manner but not IL-1alpha or IL-1beta. As expected, Sertoli cell cultures were also shown to constitutively secrete transferrin. Stimulation of these cultures with IL-1alpha, IL-1beta significantly increased their capacity to secrete transferrin. Addition of IL-1ra to unstimulated Sertoli cell cultures did not affect their capacity to secrete transferrin. Stimulation of Sertoli cell cultures with a combination of both IL-1alpha and FSH or IL-1beta and FSH showed additive effect between IL-1 and FSH in their capacity to induce transferrin secretion by these cells. However, stimulation of Sertoli cells with a combination of both IL-1ra and FSH did not affect their capacity to secrete transferrin as compared with FSH-stimulated cultures. Our results with Sertoli cells, in addition to previous data on Lydig cell and germ cells, may suggest the involvement of the IL-1 system in testicular paracrine/autocrine regulation, which could be involved in the regulation of spermatogenesis and spermiogenesis processes and male fertility.

In the present study we examined the capacity of interleukin-1 (IL-1) alpha, beta, interleukin-1 receptor antagonist (IL-1ra) and follicle stimulating hormone (FSH) to induce transferrin secretion by Sertoli cells under in vitro conditions. Primary Sertoli cell (SC) cultures from immature mice secreted constitutively transferrin. Stimulation of these cultures with IL-1alpha, IL-1beta significantly increas\d their capacity to secrete transferrin. Addition of IL-1ra to unstimulated SC cultures did not affect their capacity to secrete transferrin. Stimulation of SC cultures with a combination of both IL-1alpha and FSH or IL-1beta and FSH showed additive effect between IL-1 and FSH in their capacity to induce transferrin secretion by these cells. However, stimulation of Sertoli cells with a combination of both IL-1ra and FSH did not affect their capacity to secrete transferrin compared with FSH-stimulated cultures. Our results may suggest the involvement of testicular paracrine/autocrine factors (IL-1) and endocrine (FSH) factors in the regulation of transferrin secretion by SC. This capacity seems to be differently regulated by these factors. Thus, IL-1alpha and beta may directly affect physiological functions of the testis; which may suggest their involvement in the regulation of spermatogenesis and spermiogenesis processes and male fertility.

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. [ABSTRACT FROM AUTHOR]

Spermatogenesis is regulated mainly by endocrine factors and also by testicular paracrine/autocrine growth factors. These factors are produced by Sertoli cells, germ cells, peritubular cells and interstitial cells, mainly Leydig cells and macrophages. The interactions and the ratio between Sertoli and germ cells in the seminiferous tubules ensure successful spermatogenesis. In order to culture spermatogonial stem cells (SSCs) in vitro, researchers tried to overcome some of the obstacles - such as the low number of stem cells in the testis, absence of specific markers to identify SSCs - in addition to difficulties in keeping the SSCs alive in culture. Recently, some growth factors important for the proliferation and differentiation of SSCs were identified, such as glial cell line derived neurotrophic factor (GDNF), stem cell factor (SCF) and leukemia inhibitory factor (LIF); also, markers for SSCs at different stages were reported. Therefore, some groups succeeded in culturing SSCs (under limitations), or more differentiated cells and even were able to produce in vitro germ cells from embryonic stem cells. Thus, success in culturing SSCs is dependent on understanding the molecular mechanisms behind self-renewal and differentiation. Culture of SSCs should be a good tool for discovering new therapeutic avenue for some infertile men or for patients undergoing chemotherapy/radiotherapy (pre-puberty or post-puberty). [ABSTRACT FROM AUTHOR]

Aggressive chemotherapy treatment may lead to male infertility. Prepubertal boys do not produce sperm at this age, however, they have spermatogonial stem cells in their testes. Here, we examined the effect of intraperitoneal injection of cyclophosphamide (CP) on the capacity of immature mice (IM) to develop spermatogenesis in vivo and in vitro [using methylcellulose culture system (MCS)]. Our results show a significant decrease in testicular weight, total number of testicular cells, and the number of Sertoli, peritubular, premeiotic, and meiotic/post-meiotic cells, but an increase in the percentages of damaged seminiferous tubules in CP-treated IM compared to control. The functionality of Sertoli cells was significantly affected. The addition of testosterone to isolated cells from seminiferous tubules of CP-treated IM significantly increased the percentages of premeiotic (CD9-positive cells) and meiotic/post-meiotic cells (ACROSIN-positive cells) developed in MCS compared to control. The addition of FSH did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly decreased the percentages of CD9-positive cells and ACROSIN-positive cells. The addition of IL-1 did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly increased the percentages of VASA-positive cells and BOULE-positive cells compared to IL-1 or testosterone. Addition of TNF significantly increased only CD9-positive cells in MCS compared to control, but in combination with testosterone, it significantly decreased ACROSIN-positive cells compared to testosterone. Our results show a significant impairment of spermatogenesis in the testes of CP-treated IM, and that spermatogonial cells from these mice proliferate and differentiate to meiotic/post-meiotic cells under in vitro culture conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Spermatogenesis is the process of spermatogonial stem cell (SSC) proliferation and differentiation to generate sperm. This process is regulated by cell–cell interactions between Sertoli cells and developing SSCs by autocrine/paracrine and endocrine factors. It is also affected by cells in the interstitial compartment, such as Leydig cells and peritubular cells. Here, we demonstrate, for the first time, the presence of interleukin-34 (IL-34) in Leydig, Sertoli, and peritubular cells and in the premeiotic, meiotic, and postmeiotic cells. Its receptor, colony-stimulating factor-1 (CSF-1), has already been demonstrated in Leydig, Sertoli, premeiotic, and meiotic cells. IL-34 was detected in testicular homogenates and Sertoli cell-conditioned media, and was affected by mouse age. We showed that the addition of IL-34 in vitro to isolated cells from the seminiferous tubules of 7-day-old mice, using the methylcellulose culture system (MCS), increased the percentages and expression of the premeiotic cells (VASA), the meiotic cells (BOULE), and the meiotic/postmeiotic cells (ACROSIN) after four weeks of culture, when examined by immunofluorescence staining (IF) and qPCR analysis. It is possible to suggest that IL-34 is a novel paracrine/autocrine factor involved in the development of spermatogenesis. This factor may be used in future therapeutic strategies for the treatment of male infertility. [ABSTRACT FROM AUTHOR]

Sertoli cell-only syndrome (SCOS) affects about 26.3–57.8% of azoospermic men, with their seminiferous tubules containing only Sertoli cells. Recently, it was reported that testicular biopsies from nonobstructive azoospermic (NOA) patients contained germ cells, and that sperm could be found in the tubules of 20% of SCOS patients using testicular sperm extraction technology. Since the patients without sperm in their testicular biopsies do not have therapy to help them to father a biological child, in vitro maturation of spermatogonial stem cells (SSCs) isolated from their testis is a new approach for possible future infertility treatment. Recently, the induction of human and mice SSCs proliferation and differentiation was demonstrated using different culture systems. Our group reported the induction of spermatogonial cell proliferation and differentiation to meiotic and postmeiotic stages in mice, rhesus monkeys, and prepubertal boys with cancer using 3D agar and methylcellulose (MCS) culture systems. The aim of the study was to identify the type of spermatogenic cells present in biopsies without sperm from SCOS patients, and to examine the possibility of inducing spermatogenesis from isolated spermatogonial cells of these biopsies in vitro using 3D MCS. We used nine biopsies without sperm from SCOS patients, and the presence of spermatogenic markers was evaluated by PCR and specific immunofluorescence staining analyses. Isolated testicular cells were cultured in MCS in the presence of StemPro enriched media with different growth factors and the development of colonies/clusters was examined microscopically. We examined the presence of cells from the different stages of spermatogenesis before and after culture in MCS for 3–7 weeks. Our results indicated that these biopsies showed the presence of premeiotic markers (two to seven markers/biopsy), meiotic markers (of nine biopsies, cAMP responsive element modulator-1 (CREM-1) was detected in five, lactate dehydrogenase (LDH) in five, and BOULE in three) and postmeiotic markers (protamine was detected in six biopsies and acrosin in three). In addition, we were able to induce the development of meiotic and/or postmeiotic stages from spermatogonial cells isolated from three biopsies. Thus, our study shows for the first time the presence of meiotic and/or postmeiotic cells in biopsies without the sperm of SCOS patients. Isolated cells from some of these biopsies could be induced to meiotic and/or postmeiotic stages under in vitro culture conditions. [ABSTRACT FROM AUTHOR]