As germ cells divide and differentiate from spermatogonia to spermatozoa, they share a number of structural and functional features that are common to all generations of germ cells and these features are discussed herein. Germ cells are linked to one another by large intercellular bridges which serve to move molecules and even large organelles from the cytoplasm of one cell to another. Mitochondria take on different shapes and features and topographical arrangements to accommodate their specific needs during spermatogenesis. The nuclear envelope and pore complex also undergo extensive modifications concomitant with the development of germ cell generations. Apoptosis is an event that is normally triggered by germ cells and involves many proteins. It occurs to limit the germ cell pool and acts as a quality control mechanism. The ubiquitin pathway comprises enzymes that ubiquitinate as well as deubiquitinate target proteins and this pathway is present and functional in germ cells. Germ cells express many proteins involved in water balance and pH control as well as voltage-gated ion channel movement. In the nucleus, proteins undergo epigenetic modifications which include methylation, acetylation, and phosphorylation, with each of these modifications signaling changes in chromatin structure. Germ cells contain specialized transcription complexes that coordinate the differentiation program of spermatogenesis, and there are many male germ cell-specific differences in the components of this machinery. All of the above features of germ cells will be discussed along with the specific proteins/genes and abnormalities to fertility related to each topic.

In this review, a few well-established axioms have been challenged while others were viewed from a new perspective. The extensive literature on the blood-testis barrier has been scrutinized to help probe its mechanics and hopefully to promote understanding of the constant adaptation of the barrier function to germ cell development. Our principal conclusions are as follows: (1) Although the barrier zonule is topographically located at the base of the seminiferous epithelium it actually encircles the apex of the Sertoli cell. Consequently the long irregular processes specialized in holding and shaping the developing germ cells should be considered as apical appendages analogous to microvilli. (2) The development of the barrier zonule does not coincide with the appearance of a particular class of germ cells. (3) The barrier compartmentalizes the epithelium into only two cellular compartments: basal and lumenal. (4) Although the blood-testis barrier does sequester germ cells usually considered antigenic, immunoregulator factors other than the physical barrier seem to be involved in preventing autoimmune orchitis. (5) Structurally, a Sertoli cell junctional complex is composed of occluding, gap, close, and adhering junctions. The Sertoli cell membrane segments facing germ cells are presumably included in the continuum of the Sertoli cell junctional complex that extends all over the lateral and apical Sertoli cell membranes. (6) The modulation (i.e., formation and dismantling) of the junctions in a baso-apical direction is characteristic of the seminiferous epithelium and may be dictated by germ cell differentiation. The formation of tubulobulbar complexes and the following internalization of junction vesicles conceivably represent sequential steps of a single intricate junction elimination process that involves junction membrane segments from different cell types as part of a continual cell membrane recycling system. (7) The preferential association of junctional particles with one or the other fracture-face reflect a response to various stimuli including seasonal breeding. Changes in the affinity of the particles are generally coincidental with cytoskeletal changes. However, changes in the cytoskeleton are not necessarily accompanied by permeability changes. The number of strands seems to reflect neither the junctional permeability nor the transepithelial resistance. The diverse orientation of the strands seems to be related to the plasticity of the Sertoli cell occluding zonule. (8) Cooperation between all constituents (Sertoli cells, myoid cells, cell substratum, and germ cells) of the epithelium seems essential for the barrier zonule to function in synchrony with the germ cell differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

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The development and maintenance of the Sertoli cell junctional complex were investigated in prepubertal and adult guinea pigs. To correlate the structure of the blood-testis barrier with its permeability, the polyene antibiotic filipin (a cholesterol-binding agent of low molecular weight: 570.70) was added to the fixative as a tracer visible in freeze-fracture replicas. Discontinuous zonules, intermediate junctions (i.e., adhering fasciae) and gap junctions all proved permeable to filipin in the two age groups. Only the continuous occluding zonules characteristic of the adult guinea pig's testis were impermeable to the tracer. In pubertal animals, the establishment of the blood-testis barrier coincided with the completion of the junctional strands in occluding zonules. The formation of occluding zonules was similar in the newborn and the adult. In the adult, the Sertoli cell junctional complexes contained three types of cell junctions: occluding, adhering, and gap junctions. The sequence of occluding and adhering junctions from the base to the apex of the epithelium was the reverse of that demonstrated in most epithelia. The impermeable continuous occluding zonules at the base showed parallel patterns of uninterrupted junctional strands, whereas the permeable discontinuous zonules found higher in the epithelium showed a meandering pattern of broken strands. Our observations indicate that (1) Sertoli cell junctional complexes form near the young germinal cells at the base of the seminiferous epithelium and break down near the older germinal cells toward the apex; (2) the various patterns and orientations of the junctional strands reflect, respectively, the different stages of disintegration of the occluding zonules and the conformation of the mature Sertoli cell to the irregular contours of the germinal cells; (3) there is no relationship between permeability and junctional strand orientation; and (4) the cellular contacts between Sertoli cells and germinal cells situated below the blood-testis barrier may represent the early stages of formation of junctional elements which ultimately become incorporated into the Sertoli cell junctional complex.

Spermiogenesis is a long process whereby haploid spermatids derived from the meiotic divisions of spermatocytes undergo metamorphosis into spermatozoa. It is subdivided into distinct steps with 19 being identified in rats, 16 in mouse and 8 in humans. Spermiogenesis extends over 22.7 days in rats and 21.6 days in humans. In this part, we review several key events that take place during the development of spermatids from a structural and functional point of view. During early spermiogenesis, the Golgi apparatus forms the acrosome, a lysosome-like membrane bound organelle involved in fertilization. The endoplasmic reticulum undergoes several topographical and structural modifications including the formation of the radial body and annulate lamellae. The chromatoid body is fully developed and undergoes structural and functional modifications at this time. It is suspected to be involved in RNA storing and processing. The shape of the spermatid head undergoes extensive structural changes that are species-specific, and the nuclear chromatin becomes compacted to accommodate the stream-lined appearance of the sperm head. Microtubules become organized to form a curtain or manchette that associates with spermatids at specific steps of their development. It is involved in maintenance of the sperm head shape and trafficking of proteins in the spermatid cytoplasm. During spermiogenesis, many genes/proteins have been implicated in the diverse dynamic events occurring at this time of development of germ cells and the absence of some of these have been shown to result in subfertility or infertility.

The gene for proprotein convertase subtilisin/kexin-like 4 (PCSK4, previously known as PC4) is primarily transcribed in testicular spermatogenic cells. Its inactivation in mouse causes severe male subfertility. To better understand the reproductive function of PCSK4, we examined its subcellular localization in the testicular epithelium via immunohistochemistry, and on intact sperm via indirect immunofluorescence and immunoelectron microscopy. PCSK4 was detected in the acrosomal granules of round spermatids, in the acrosomal ridges of elongated spermatids, and on the sperm plasma membrane overlying the acrosome. We also investigated PCSK4 relevance for sperm acquisition of fertilizing ability by comparing wild-type and PCSK4-null sperm for their abilities in capacitation, acrosome reaction, and egg binding in vitro. PCSK4-null sperm underwent capacitation at a faster rate; they were induced to acrosome react by lower concentrations of zona pellucida; and their egg-binding ability was only half that of wild-type sperm. These sperm physiologic anomalies likely contribute to the severe subfertility of PCSK4-deficient male mice.

Gap junction-mediated communication helps synchronize interconnected Sertoli cell activities. Besides, coordination of germ cell and Sertoli cell activities depends on gap junction-mediated Sertoli cell-germ cell communication. This report assesses mechanisms underlying the regulation of connexin 46 (Cx46) and Cx50 in mouse testis and those accompanying a "natural" seasonal and a pathological arrest of spermatogenesis, resulting from autoimmune orchitis (AIO) in mink. Furthermore, the impact of deleting Cx46 or Cx50 on the expression, phosphorylation of junction proteins, and spermatogenesis is evaluated. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. Cx46 mRNA and protein expression increased, whereas Cx50 decreased with adulthood in normal mice and mink. During the mink active spermatogenic phase, Cx50 became phosphorylated and localized to the site of the blood-testis barrier. By contrast, Cx46 was dephosphorylated and associated with annular junctions, suggesting phosphorylation/dephosphorylation of Cx46 and Cx50 involvement in the barrier dynamics. Cx46-positive annular junctions in contact with lipid droplets were found. Cx46 and Cx50 expression and localization were altered in mink with AIO. The deletion of Cx46 or Cx50 impacted on other connexin expression and phosphorylation and differently affected tight and adhering junction protein expression. The level of apoptosis, determined by ELISA, and a number of Apostain-labeled spermatocytes and spermatids/tubules were higher in mice lacking Cx46 (Cx46-/-) than wild-type and Cx50-/- mice, arguing for life-sustaining Cx46 gap junction-mediated exchanges in late-stage germ cells secluded from the blood by the barrier. The data show that expression and phosphorylation of Cx46 and Cx50 are complementary in seminiferous tubules.

Because in mammals the anterior pituitary lacks innervation, we investigated whether gap junctions established between selected cells within the gland are part of an intrapituitary mechanism to ensure physiological synchronization of cells involved in the control of hormone secretion. We report here the dynamics of anterior pituitary connexin 43 (Cx43)-gap junctions throughout the mink (Mustela vison) annual reproductive cycle and its relationship with the anterior pituitary prolactin (PRL) content that parallels variations in serum PRL levels documented in the literature. We found that PRL anterior pituitary levels were maximal in spring and during lactation and that they were minimal in autumn and winter. Anterior pituitary Cx43 levels were maximal during periods of high PRL secretion. During these periods, Cx43-positive gap junctions localized to stellate-shaped cells occupying the center of anterior pituitary follicles and to the rounded cells occupying the remaining follicles. Connexin 43-positive gap junctions were also observed between adjacent follicles. During periods of low PRL pituitary content, Cx43-positive gap junctions localized to the stellate cells but not to the cells of the remaining follicles. Moreover, Cx43 labeling was undetected between adjacent follicles. To assess between which cells within the mink anterior pituitary the Cx43 gap junctions were established, the different anterior pituitary cell populations were separated by a discontinuous Percoll gradient, and Western blot analyses of each cell population using Cx43 antibodies were performed. The immunoblots showed a Cx43 immunoreactive band associated with the cell layer enriched in S-100-positive, stellate-shaped cells. The result was confirmed by fluorescence microscopy studies that showed that Cx43-mediated gap junctions were established preferentially between the cultured S-100-positive, elongated cells. The results show that in mink stellate cells, the junctional machinery associated with the Cx43 protein varies in synchrony with the anterior pituitary PRL content throughout the mink annual reproductive cycle. It is suggested that the Cx43 gap junctions on the stellate cells play an important role in the synchronization of cellular activity within selected follicles of the anterior pituitary, thus contributing to the control of PRL secretion during the annual reproductive cycle.