Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation

The niche is considered to play an important role in stem cell biology. Sertoli cells are the only somatic cells in the seminiferous tubule that closely interact with germ cells to create a favorable environment for spermatogenesis. However, little is known about how Sertoli cells develop to form the male germ line niche. We report here that Sertoli cells recovered and dissociated from testes of donor male mice can be microinjected into recipient testes, form mature seminiferous tubule structures, and support spermatogenesis. Sertoli cells from perinatal donors had a dramatically greater capacity for generating seminiferous tubules than those from adult donors. Furthermore, transplantation of wild-type Sertoli cells into infertile Steel/Steel(dickie) testes created a permissive testicular microenvironment for generating spermatogenesis and spermatozoa. Thus, our results demonstrate that the male germ line stem cell niche can be transferred between animals. In addition, the technique provides a novel tool with which to analyze spermatogenesis and might provide a mechanism for correcting fertility in males suffering from supporting cell defects.     

An ultrastructural study of the effect of a diabetogenic dose of alloxan on the secretory ameloblasts of the rat incisor

Sertoli cells of the ground squirrel (Spermophilus lateralis), a seasonal breeder, were examined by light and electron microscopy and their structure, particularly the organization of the cytoskeleton, was related to events that occur in the seminiferous epithelium during spermatogenesis. Among the events considered and described are the apical movement of elongate spermatids, withdrawal of residual cytoplasm from germ cells, transport of smooth endoplasmic reticulum (SER) between the base and apex of the Sertoli cells, and sperm release. These events are dramatically evident in this species because the seminiferous epithelium is thin, i.e., there are few germ cells, and both the germ cells and Sertoli cells are large. Sertoli cells of the ground squirrel have a remarkably well developed cytoskeleton. Microfilaments occur throughout the cell but are most evident in ectoplasmic specializations associated with junctions. Intermediate filaments occur around the nucleus, as a layer at the base of the cell, and adjacent to desmosome-like junctions with germ cells. Intermediate filaments, together with microtubules, are also abundant in regions of the cell involved with the transport of SER, in cytoplasm associated with elongate spermatids, and in processes that extend into the residual cytoplasm of germ cells. Our observations of ultrastructure are consistent with the hypothesis that Sertoli cell microtubules are involved with the movement of germ cells within the seminiferous epithelium, and further implicate these structures as possibly playing a role in the retraction of residual cytoplasm from germ cells and the intracellular transport of SER. The abundance and organization of intermediate filaments suggest that these cytoskeletal elements may also be involved with events that occur during spermatogenesis.     

Arylsulfatase A deficiency causes seminolipid accumulation and a lysosomal storage disorder in Sertoli cells

Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of male germ cells. During spermatogenesis, apoptosis occurs in >50% of total germ cells. Sertoli cells phagocytose these apoptotic germ cells and degrade their components using lysosomal enzymes. Here we demonstrated that SGG was a physiological substrate of Sertoli lysosomal arylsulfatase A (ARSA). SGG accumulated in Sertoli cells of Arsa(-/-) mice, and at 8 months of age, this buildup led to lysosomal swelling and other cellular abnormalities typical of a lysosomal storage disorder. This disorder likely compromised Sertoli cell functions, manifesting as impaired spermatogenesis and production of sperm with near-zero fertilizing ability in vitro. Fecundity of Arsa(-/-) males was thus reduced when they were older than 5 months. Sperm SGG is known for its roles in fertilization. Therefore, the minimal sperm fertilizing ability of 8-month-old Arsa(-/-) males may be explained by the 50% reduction of their sperm SGG levels, a result that was also observed in testicular germ cells. These unexpected decreases in SGG levels might be partly due to depletion of the backbone lipid palmitylpalmitoylglycerol that is generated from the SGG degradation pathway in Sertoli cells and normally recycled to new generations of primary spermatocytes for SGG synthesis.     

Normal fertility in male mice with deletion of β‐catenin gene in germ cells

As a dual function protein, β‐catenin affects both cell adhesion and mediates canonical Wnt/β‐catenin cell signaling. β‐Catenin is prominently expressed in somatic Sertoli cells in the testis and postmeiotic germ cells, suggesting an additional role in spermatogenesis. It was reported previously that Cre/loxP‐mediated conditional inactivation of the β‐catenin gene (Ctnnb1) in male gonads using a protamine promoter‐driven Cre transgene (Prm‐cre) resulted in partial infertility, reduced sperm count, and abnormal spermatogenesis. In this report, we demonstrated that the conditional deletion of Ctnnb1 using a germ cell specific Cre transgene (Stra8‐icre) had no effect on male fertility. We have shown that the Stra8‐icre transgene was highly efficient in generating deletion in early pre‐meiotic and post‐meiotic cells. No differences in anatomical or histological presentation were found in the mutant testis, the production of viable sperm was similar, and no abnormalities in DNA sperm content were detected. We concluded that β‐catenin is fully dispensable in germ cells for spermatogenesis. The conflicting results from the earlier study may have been due to off‐target expression of Prm‐cre in testicular somatic cells. In future studies, the analysis of conditional mutants using several Cre‐transgenes should be encouraged to reduce potential errors. genesis 52:328–332, 2014. © 2014 Wiley Periodicals, Inc.     

Polarity proteins and actin regulatory proteins are unlikely partners that regulate cell adhesion in the seminiferous epithelium during spermatogenesis

In mammalian testis, spermatogenesis takes place in the seminiferous epithelium of the seminiferous tubule, which is composed of a series of cellular events. These include: (i) spermatogonial stem cell (SSC) renewal via mitosis and differentiation of SSC to spermatogenia, (ii) meiosis, (iii) spermiogenesis, and (iv) spermiation. Throughout these events, developing germ cells remain adhered to the Sertoli cell in the seminiferous epithelium amidst extensive cellular, biochemical, molecular and morphological changes to obtain structural support and nourishment. These events are coordinated via signal transduction at the cell-cell interface through cell junctions, illustrating the significance of cell junctions and adhesion in spermatogenesis. Additionally, developing germ cells migrate progressively across the seminiferous epithelium from the stem cell niche, which is located in the basal compartment near the basement membrane of the tunica propria adjacent to the interstitium. Recent studies have shown that some apparently unrelated proteins, such as polarity proteins and actin regulatory proteins, are in fact working in concert and synergistically to coordinate the continuous cyclic changes of adhesion at the Sertoli-Sertoli and Sertoli-germ cell interface in the seminiferous epithelium during the epithelial cycle of spermatogenesis, such that developing germ cells remain attached to the Sertoli cell in the epithelium while they alter in cell shape and migrate across the epithelium. In this review, we highlight the physiological significance of endocytic vesicle-mediated protein trafficking events under the influence of polarity and actin regulatory proteins in conferring cyclic events of cell adhesion and de-adhesion. Furthermore, these recent findings have unraveled some unexpected molecules to be targeted for male contraceptive development, which are also targets of toxicant-induced male reproductive dysfunction.     

Seasonal spermatogenic cycle and morphology of germ cells in the viviparous lizard Mabuya brachypoda (Squamata,Scincidae)

We describe seasonal variations of the histology of the seminiferous tubules and efferent ducts of the tropical, viviparous skink, Mabuya brachypoda, throughout the year. The specimens were collected monthly, in Nacajuca, Tabasco state, Mexico. The results revealed strong annual variations in testicular volume, stages of the germ cells, and diameter and height of the epithelia of seminiferous tubules and efferent ducts. Recrudescence was detected from November to December, when initial mitotic activity of spermatogonia in the seminiferous tubules were observed, coinciding with the decrease of temperature, photoperiod and rainy season. From January to February, early spermatogenesis continued and early primary and secondary spermatocytes were developing within the seminiferous epithelium. From March through April, numerous spermatids in metamorphosis were observed. Spermiogenesis was completed from May through July, which coincided with an increase in temperature, photoperiod, and rainfall. Regression occurred from August through September when testicular volume and spermatogenic activity decreased. During this time, the seminiferous epithelium decreased in thickness, and germ cell recruitment ceased, only Sertoli cells and spermatogonia were present in the epithelium. Throughout testicular regression spermatocytes and spermatids disappeared and the presence of cellular debris, and scattered spermatozoa were observed in the lumen. The regressed testes presented the total suspension of spermatogenesis. During October, the seminiferous tubules contained only spermatogonia and Sertoli cells, and the size of the lumen was reduced, giving the appearance that it was occluded. In concert with testis development, the efferent ducts were packed with spermatozoa from May through August. The epididymis was devoid of spermatozoa by September. M. brachypoda exhibited a prenuptial pattern, in which spermatogenesis preceded the mating season. The seasonal cycle variations of spermatogenesis in M. brachypoda are the result of a single extended spermiation event, which is characteristic of reptilian species. J. Morphol. © 2012 Wiley Periodicals, Inc.     

The Sertoli cell in vivo and in vitro

The Sertoli cell extends from the basement membrane of the seminiferous tubule towards its lumen; it sends cytoplasmic processes which envelop different generations of germ cells. The use of Sertoli cell culture began to develop in 1975. To reduce germ cell contamination immature animals are generally used as Sertoli cell donors. Sertoli cell mitosis essentially occurs in sexually immature testes in mammals; mitosis of these cells is observed in vitro during a limited period of time. Sertoli cells in vivo perform an impressive range of functions: structural support of the seminiferous epithelium, displacement of germ cells and release of sperm; formation of the Sertoli cell blood-testis barrier; secretion of factors and nutrition of germ cells; phagocytosis of degenerating germ cells and of germ cell materials. Some of the Sertoli cell functions can be studied in vitro. The recent development of Sertoli cell culture on permeable supports (with or without extracellular matrix) has resulted in progress in understanding the vectorial secretion of several Sertoli cell markers. In addition to FSH and testosterone, several other humoral factors are known to influence Sertoli cell function. Furthermore, myoid cells bordering the tubules as well as germ cells are capable of regulating Sertoli cell activity. Sertoli cells are the most widely used testicular cells for in vitro toxicology. The testis is highly vulnerable to xenobiotics and radiations, yet the number of studies undertaken in this field is insufficient and should be drastically increased.     

Long-term vitamin A deficiency induces alteration of adult mouse spermatogenesis and spermatogonial differentiation: direct effect on spermatogonial gene expression and indirect effects via somatic cells

The objective of this study was to further understand the genetic mechanisms of vitamin A deficiency (VAD) induced arrest of spermatogonial stem-cell differentiation.Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that leads to the formation of sperm in the seminiferous epithelium. VAD causes early cessation of spermatogenesis, characterized by degeneration of meiotic germ cells, leading to seminiferous tubules containing mostly type A spermatogonia and Sertoli cells. These observations led us to the hypothesis that VAD affects not only germ cells but also somatic cells.To investigate the effects of VAD on spermatogenesis in mice we used adult Balb/C mice fed with Control or VAD diet for an extended period of time (6–28 weeks). We first observed the chronology, then the extent of the effects of VAD on the testes. Using microarray analysis of isolated pure populations of spermatogonia, Leydig and Sertoli cells from control and VAD 18- and 25-week mice, we examined the effects of VAD on gene expression and identified target genes involved in the arrest of spermatogonial differentiation and spermatogenesis.Our results provide a more precise definition of the chronology and magnitude of the consequences of VAD on mouse testes than the previously available literature and highlight direct and indirect (via somatic cells) effects of VAD on germ cell differentiation.     

BNC1 is required for maintaining mouse spermatogenesis

Basonuclin (BNC1) is a zinc finger protein expressed primarily in gametogenic cells and proliferative keratinocytes. Our previous work suggested that BNC1 is present in spermatogonia, spermatocytes, and spermatids, but absent in the Sertoli cells. BNC1's role in spermatogenesis is unknown. Here, we show that BNC1 is required for the maintenance of spermatogenesis. Bnc1-null male mice were sub-fertile, losing germ cells progressively with age. The Bnc1-null seminiferous epithelia began to degenerate before 8 weeks of age and eventually became Sertoli cell-only. Sperm count and motility also declined with age. Furthermore, Bnc1 heterozygotes, although fertile, showed a significant drop in sperm count and in testis weight by 24 weeks of age, suggesting a dosage effect of Bnc1 on testis development. In conclusion, our data demonstrate for the first time BNC1's essential role in maintaining mouse spermatogenesis.     

Cytokines and junction restructuring events during spermatogenesis in the testis: An emerging concept of regulation

During spermatogenesis in mammalian testes, junction restructuring takes place at the Sertoli–Sertoli and Sertoli–germ cell interface, which is coupled with germ cell development, such as cell cycle progression, and translocation of the germ cell within the seminiferous epithelium. In the rat testis, restructuring of the blood–testis barrier (BTB) formed between Sertoli cells near the basement membrane and disruption of the apical ectoplasmic specialization (apical ES) between Sertoli cells and fully developed spermatids (spermatozoa) at the luminal edge of the seminiferous epithelium occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis. These two processes are essential for the translocation of primary spermatocytes from the basal to the apical compartment to prepare for meiosis, and the release of spermatozoa into the lumen of the seminiferous epithelium at spermiation, respectively. Cytokines, such as TNFα and TGFβ3, are present at high levels in the microenvironment of the epithelium at this stage of the epithelial cycle. Since these cytokines were shown to disrupt the BTB integrity and germ cell adhesion, it was proposed that some cytokines released from germ cells, particularly primary spermatocytes, and Sertoli cells, would induce restructuring of the BTB and apical ES at stage VIII of the seminiferous epithelial cycle. In this review, the intricate role of cytokines and testosterone to regulate the transit of primary spermatocytes at the BTB and spermiation will be discussed. Possible regulators that mediate cytokine-induced junction restructuring, including gap junction and extracellular matrix, and the role of testosterone on junction dynamics in the testis will also be discussed.     

Taenia crassiceps: A secretion-substance of low molecular weight leads to disruption and apoptosis of seminiferous epithelium cells in male mice

The present research was performed to isolate and study the effects of a low molecular weight (<1300 Da) parasite-associated substance, obtained from peritoneal fluids of female mice infected with Taenia crassiceps cysticerci, on seminiferous epithelium cells of male mice testis. The results showed an intense disruption of Sertoli cells and germ cells within the seminiferous tubules of experimental mice, along with the destruction of their gap junction (GJ). Significant generalized apoptosis of germ cells within seminiferous tubules was determined by TUNEL staining (P = 0.0159). In addition, a significant number of infiltrating macrophages were found in the luminal space of these seminiferous tubules (P < 0.0001). Finally, electron microscopy studies revealed structural and morphological abnormalities in the somatic cells (Sertoli and Leydig cells) and in the germ cells, primarily in the round and elongate spermatids.     

Influence of hormonal imbalance on the integrity of seminiferous epithelium in the testes of adult rats chronically exposed to letrozole and rats exposed to soya isoflavones during the prenatal period,lactation, and up to sexual maturity

The structural integrity of the germ cells in the seminiferous epithelium and the correct process of spermatogenesis are made possible by proteins that participate in the formation of different types of junctions. This study was performed on samples of the testes of 4 groups (2 experimental and 2 corresponding control) of male Wistar rats. In the first experimental group, the adult rats received letrozole – a nonsteroidal inhibitor of cytochrome P450 aromatase (P450arom). The second experimental group was exposed to soya isoflavones during the prenatal period, lactation, and up to sexual maturity. The aim of this study was to examine the immunoexpression of β-catenin, N-cadherin, occludin, connexin43, annexin V, and advanced glycation end products (AGE) in the seminiferous epithelium of rat testes with chronic estrogen deficiency and of rats exposed to soya isoflavones. Series of sections of the testes were stained using PAS and silver impregnation. Moreover, immunohistochemistry tests were performed. A semi-quantitative determination of protein immunoexpression was performed using Image J. The number of annexin V positive Sertoli cells per tubule were counted manually. Comparisons between the experimental and corresponding control groups were performed using a non-parametric Mann-Whitney U test. The most common alterations were prematurely sloughed germ cells in the lumen of the seminiferous tubules and invaginations of the seminiferous tubules. We observed a lower number of annexin V positive Sertoli cells and a lower expression of N-cadherin and occludin in the seminiferous epithelium of both groups of rats with hormonal imbalances. Moreover, a higher expression of AGE, a lower expression of connexin 43 and a lower amount of reticular fibers in the basal lamina of seminiferous tubules was present in rats treated with letrozole and a higher expression of β-catenin was found in rats exposed to soya isoflavones. The hormonal imbalance between androgens and estrogens resulted in a decreased number of annexin V positive Sertoli cells. This may be associated with a failed clearance of apoptotic germ cells that leads to disturbances in the blood-testis-barrier (BTB) by affecting the expression of junctional proteins in the seminiferous epithelium. Moreover, a decreased level of estrogens was also associated with an increased expression of AGEs and with a changed composition of basal lamina in the seminiferous tubules of rats. These changes could lead to germ cell sloughing and invaginations of the seminiferous tubules.     

Distribution of gelsolin in human testis

Gelsolin, an actin-binding and severing protein present in many mammalian cells, was characterized in human testis. Although abundant in testicular extracts, gelsolin was not detected in purified spermatogenic cells by immunoblot analysis. Immunofluorescence studies of testis sections showed that gelsolin has two main localizations: peritubular cells and the seminiferous epithelium. In peritubular cells, gelsolin was present together with α-SM actin, in agreement with the myoid cell characteristics of these cells. In a large proportion of the tubules, gelsolin was found mainly, together with actin, in the apical part of the seminiferous epithelium. This localization of gelsolin also was observed in seminiferous tubules with a partial or complete absence of germinal cells, which evokes a presence of gelsolin at the apex of Sertoli cells. However, in normal testis, a complex pattern of gelsolin labeling was also present, mostly in the apical third of the epithelium, around cells or groups of cells, mainly spermatids, and, less frequently, in various other localizations from the apical to the basal part of the seminiferous epithelium. Taken together, these observations suggest that gelsolin may play different functions in the seminiferous epithelium: (1) regulation of the dynamic alterations of the actin cytoskeleton in the apical cytoplasm of Sertoli cells, and (2) modification of actin filaments assemblies in specific structures at germ cell-Sertoli cell contacts. Thereby, the actin-modulating properties of gelsolin are probably involved in reorganization of the seminiferous epithelium related to germ cell differentiation. Mol. Reprod. Dev. 48:63–70, 1997. © 1997 Wiley-Liss, Inc.     

Ultrastructure of sustentacular (Sertoli) cells in the bovine testis

In the present communication, ultrastructural and cytochemical aspects of mature bovine Sertoli cells and their relationship to the different stages of germ cell development are described. As in other mammalian species, different types of junctional specializations exist between Sertoli and germ cells in the bovine seminiferous epithelium, including desmosome-like junctions, Sertoli cell ectoplasmic specializations and tubulobulbar complexes. The functional significance of the morphological results and the interactions of Sertoli and germ cells during spermatogenesis are discussed.     

MicroRNA参与调控睾丸支持细胞的增殖与粘附功能

精子发生过程需要生精细胞及睾丸体细胞的共同参与,这两种细胞也决定着睾丸的发育及雄性生育力。支持细胞是生精小管中唯一的体细胞,在正常精子发生过程中发挥重要的作用。支持细胞增殖与粘附功能的异常将导致精子发生异常,进而引发雄性不育。近年来研究发现,microRNA (miRNA)可调控支持细胞的增殖与粘附功能,其表达水平在激素、内分泌干扰素和营养状况等多种因素作用下发生特异性变化。本文总结了与睾丸支持细胞增殖与粘附功能相关的miRNA及其作用机制,以期发现并鉴定更多与支持细胞相关的miRNA,进而为探索与支持细胞相关不育症的病因提供理论依据。     

Factors affecting spermatogenesis in the stallion

Spermatogenesis is a process of division and differentiation by which spermatozoa are produced in seminiferous tubules. Seminiferous tubules are composed of somatic cells (myoid cells and Sertoli cells) and germ cells (spermatogonia, spermatocytes, and spermatids). Activities of these three germ cells divide spermatogenesis into spermatocytogenesis, meiosis, and spermiogenesis, respectively. Spermatocytogenesis involves mitotic cell division to increase the yield of spermatogenesis and to produce stem cells and primary spermatocytes. Meiosis involves duplication and exchange of genetic material and two cell divisions that reduce the chromosome number to haploid and yield four spermatids. Spermiogenesis is the differentiation without division of spherical spermatids into mature spermatids which are released from the luminal free surface as spermatozoa. The spermatogenic cycle (12.2 days in the horse) is superimposed on the three major divisions of spermatogenesis which takes 57 days. Spermatogenesis and germ cell degeneration can be quantified from numbers of germ cells in various steps of development throughout spermatogenesis, and quantitative measures are related to number of spermatozoa in the ejaculate. Germ cell degeneration occurs throughout spermatogenesis; however, the greatest seasonal impact on horses occurs during spermatocytogenesis. Daily spermatozoan production is related to the amount of germ cell degeneration, pubertal development, season of the year, and aging. Number of Sertoli cells and amount of smooth endoplasmic reticulum of Leydig cells and Leydig cell number are related to spermatozoan production. Seminiferous epithelium is sensitive to elevated temperature, dietary deficiencies, androgenic drugs (anabolic steroids), metals (cadmium and lead), x-ray exposure, dioxin, alcohol, and infectious diseases. However, these different factors may elicit the same temporary or permanent response in that degenerating germ cells become more common, multinucleate giant germ cells form by coalescence of spermatocytes or spermatids, the ratio of germ cells to Sertoli cells is reduced, and spermatozoan production is adversely affected. In short, spermatogenesis involves both mitotic and meiotic cell divisions and an unsurpassed example of cell differentiation in the production of the spermatozoon. Several extrinsic factors can influence spermatogenesis to cause a similar degenerative response of the seminiferous epithelium and reduce fertility of stallions.     

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.