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Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP and EGF pathways are used to mediate soma-germline communication. Here, we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germ line. When gap junctions between the soma and germ line are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg (Inx4) and Inx2 on the germ line and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germ line to ensure proper spermatogenesis in Drosophila. Moreover, we show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues., (© 2015. Published by The Company of Biologists Ltd.)

Background: During late prenatal and early postnatal life, the reproductive system in males undergoes an extensive series of physiological and morphological changes. Prenatal ethanol (EtOH) exposure has marked effects on the development of the reproductive system, with long-term effects on function in adulthood. The present study tested the hypothesis that prenatal EtOH exposure will delay the onset of spermatogenesis., Methods: Development of the seminiferous tubules and the onset of spermatogenesis were examined utilizing a rat model of fetal alcohol spectrum disorder (FASD). Male offspring from ad libitum-fed control (C), pair-fed (PF), and EtOH-fed (prenatal alcohol exposure [PAE]) dams were terminated on postnatal (PN) days 5, 15, 18, 20, 25, 35, 45, and 55, to investigate morphological changes through morphometric analysis of the testes from early neonatal life through young adulthood., Results: PAE males had lower relative (adjusted for body weight) testis weights compared with PF and/or C males from PN15 through puberty (PN45). In addition, fewer gonocytes (primordial germ cells) were located on the basal lamina on PN5, while more of those touching the basal lamina were dividing in PAE compared with PF and C males, suggesting delayed cell division and migration processes. As well, the percentage of tubules with open lumena was lower in PAE compared with PF and C males on PN18 and 20, and PAE males had fewer primary spermatocytes per tubule on PN18 and round spermatids per tubule on PN25 compared with C males. Finally, the percentage of tubules at stages VII and VIII, when mature spermatids move to the apex of the epithelium and are released, was lower in PAE compared with PF and/or C males in young adulthood (PN55)., Conclusions: Maternal EtOH consumption appears to delay both reproductive development and the onset of spermatogenesis in male offspring, with effects persisting at least until young adulthood., (Copyright © 2013 by the Research Society on Alcoholism.)

Human male infertility affects approximately 5% of men, with one-third suffering from testicular failure, likely the result of an underlying genetic abnormality that disrupts spermatogenesis during development. Mouse models of male infertility such as the Ppp1cc knockout mouse display very similar phenotypes to humans with testicular failure. Male Ppp1cc mutant mice are sterile due to disruptions in spermatogenesis that begin during prepubertal testicular development, and continue into adulthood, often resulting in loss of germ cells to the point of Sertoli cell-only syndrome. The current study employs light and electron microscopy to identify new morphological abnormalities in Ppp1cc mutant seminiferous epithelium. This study reveals that germ cells become delayed in their development around stages VII and VIII of spermatogenesis. Loss of these cells likely results in the reduced numbers of elongating spermatids and spermatozoa previously observed in mutant animals. Interestingly, Ppp1cc mutants also display reduced numbers of spermatogonia compared with their wild-type counterparts. Using electron microscopy, we have shown that junction complexes in Ppp1cc mutants are ultrastructurally normal, and therefore do not contribute to the breakdown in tissue architecture seen in mutants. Electron microscopy revealed major acrosomal and chromatin condensation defects in Ppp1cc mutants. Our observations are discussed in the context of known molecular changes in Ppp1cc mutant testes.

The cytoskeleton of terminally differentiated mammalian Sertoli cells is one of the most elaborate of those that have been described for cells in tissues. Actin filaments, intermediate filaments and microtubules have distinct patterns of distribution that change during the cyclic process of spermatogenesis. Each of the three major cytoskeletal elements is either concentrated at or related in part to intercellular junctions. Actin filaments are concentrated in unique structures termed ectoplasmic specializations that function in intercellular adhesion, and at tubulobulbar complexes that are thought to be involved with junction internalization during sperm release and movement of spermatocytes through basal junctions between neighboring Sertoi cells. Intermediate filaments occur in a perinuclear network which has peripheral extensions to desmosome-like junctions with adjacent cells and to small hemidesmosome-like attachments to the basal lamina. Unlike in most other epithelia where the intermediate filaments are of the keratin type, intermediate filaments in mature Sertoli cells are of the vimentin type. The function of intermediate filaments in Sertoli cells in not entirely clear; however, the pattern of filament distribution and the limited experimental data available are consistent with a role in maintaining tissue integrity when the epithelium is mechanically stressed. Microtubules are abundant in Sertoli cells and are predominantly oriented parallel to the long axis of the cell. Microtubules are involved with maintaining the columnar shape of Sertoli cells, with transporting and positioning organelles in the cytoplasm, and with secreting seminiferous tubule fluid. In addition, microtubule-based transport machinery is coupled to intercellular junctions to translocate and position adjacent spermatids in the epithelium. Although the cytoskeleton of Sertoli cells has structural and functional properties common to cells generally, there are a number of properties that are unique and that appear related to processes fundamental to spermatogenesis and to interfacing somatic cells both with similar neighboring somatic cells and with differentiating cells of the germ cell line.

Tubulobulbar complexes may be part of the mechanism by which intercellular adhesion junctions are internalized by Sertoli cells during sperm release. These complexes develop in regions where Sertoli cells are attached to adjacent cells by intercellular adhesion junctions termed ectoplasmic specializations. At sites where Sertoli cells are attached to spermatid heads, tubulobulbar complexes consist of fingerlike processes of the spermatid plasma membrane, corresponding invaginations of the Sertoli cell plasma membrane, and a surrounding cuff of modified Sertoli cell cytoplasm. At the terminal ends of the complexes occur clusters of vesicles. Here we show that tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations and that the structures share similar molecular components. In addition, the adhesion molecules nectin 2 and nectin 3, found in the Sertoli cell and spermatid plasma membranes, respectively, are concentrated at the distal ends of tubulobulbar complexes. We also demonstrate that double membrane bounded vesicles are associated with the ends of tubulobulbar complexes and nectin 3 is present on spermatids, but is absent from spermatozoa released from the epithelium. These results are consistent with the conclusion that Sertoli cell and spermatid membrane adhesion domains are internalized together by tubulobulbar complexes. PKCalpha, a kinase associated with endocytosis of adhesion domains in other systems, is concentrated at tubulobulbar complexes, and antibodies to endosomal and lysosomal (LAMP1, SGP1) markers label the cluster of vesicles associated with the ends of tubulobulbar complexes. Our results are consistent with the conclusion that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations and with the internalization of intercellular membrane adhesion domains during sperm release.

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.

I have studied the distribution of microtubules in rat Sertoli cells during spermatogenesis. The distribution of microtubules was determined by indirect immunofluorescence in fragments of seminiferous epithelium which were mechanically dissociated from perfusion-fixed testes. The presence of microtubules in regions indicated by immunofluorescence was confirmed with electron microscopy. Microtubules are not evident in Sertoli cell regions surrounding early spermatogenic cells, but are abundant in cytoplasm adjacent to apical crypts containing elongate spermatids. In regions surrounding crypts, microtubules are uniformly aligned parallel to the long axis of Sertoli cells. Microtubules in these more central, or columnar, regions of the cells extend from supranuclear levels to the apex of the epithelium. In apical processes surrounding late spermatids, microtubules conform to the contours of the hook-shaped spermatid heads. These microtubules appear continuous with those in the columnar regions of the Sertoli cells. Changes in microtubule distribution observed in the rat are generally similar to, but less elaborate than, those reported previously in the squirrel. These changes may be characteristic of mammalian spermatogenesis in general.

Two types of filaments were observed within the subacrosomal space of rat spermatids. The first of these types was characterized as actin by demonstration of actin filament affinity for myosin S-1 subfragments. Actin filaments were noted in the subacrosomal space shortly after the acrosomal sac made contact with the nucleus. As the acrosome increased its surface area contact with the spermatid nucleus, the number of layers of subacrosomal filaments increased. Pre-treatment with detergent, which in addition to permeablizing cells to allow entry of S-1, also caused the acrosome to vesiculate and the subacrosomal space to widen. In such preparations filaments were more easily visualized and appeared to extend between the nuclear and acrosomal membranes, indicating, but not proving, attachment to these membranes. During spermatid clongation, the number of actin filaments in the subacrosomal space increased greatly, especially over the dorsal convex region of the spermatid head. The polarity of the majority of filaments was not ascertainable since filaments were tightly packed within the narrow subacrosomal space. In late spermiogenesis (steps 18 and 19), actin filaments were no longer detected within the subacrosomal space. A second and much thicker type of filamentous structure was observed in the subacrosomal space of spermatids at steps 14-17 of spermiogenesis. About 14 nm in diameter (10-15 nm measurement range depending on fixation protocol utilized), these filaments did not decorate with myosin S-1 subfragments and were found in subacrosomal regions not containing actin. Fourteen nanometer filaments were seen in parallel array along the ventral folded portion of the nuclear membrane and extended partially around the nucleus. Like actin filaments. 14 nm filaments were not seen in the subacrosomal space during late spermiogenesis.

We have studied the distribution of microtubules and intermediate filaments in mammalian Sertoli cells during spermatogenesis. The arrangement of microtubules was determined, by indirect immunofluorescence, in ground squirrel testes that were 1) fixed, mechanically fragmented, and attached to polylysine-coated slides, and 2) fixed, embedded in polyethylene glycol, and sectioned. Intermediate filament patterns were determined, also by indirect immunofluorescence, in sections of unfixed rat testis. Results from these studies were confirmed and extended using electron microscopy. Microtubules first become evident in lateral processes that embrace round spermatids. When spermatids elongate and become situated in apical crypts of Sertoli cells, the microtubules become oriented parallel to the long axis of Sertoli cells and surround the crypts. As spermatids mature and acquire a saucer shape, apical microtubules progressively concentrate in Sertoli cell regions adjacent to the acrosome and eventually form discrete C-shaped structures that disappear during spermiation. Intermediate filaments in rat Sertoli cells are centered around the nucleus. From perinuclear regions, filaments extend toward desmosome-like junctions with early spermatogenic cells and into the apical cytoplasm where they have a transient association with crypts containing elongate spermatids. Filaments amongst crypts are most evident in early stages of the spermatogenic cycle when apical crypts are situated deep within the epithelium. They become less evident and eventually disappear as spermatids assume a more apical position. Our fluorescence studies and ultrastructural analyses indicate that the association of intermediate filaments with crypts is specific to regions adjacent to the dorsal or convex aspect of spermatid heads. In these regions, approximately 8 to 12 uniformly aligned filaments are intimately associated with actin filaments in ectoplasmic specializations surrounding the crypts. We conclude that, like actin, the distribution of microtubules and intermediate filaments changes in Sertoli cells during spermatogenesis. The distribution of microtubules correlates with the irregular columnar shape of Sertoli cells. We suspect that the apically situated intermediate filaments may play a role in anchoring or positioning Sertoli cell crypts deep within the epithelium during the early stages of the spermatogenic cycle.