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Spermatogenesis is a multifaceted and meticulously orchestrated process involving meiosis, chromatin build up, transcriptional and translational hushing, and spermiogenesis. Male germ cell lines GC-1spg (GC-1) and GC-2(spd)ts (GC-2) provide a useful resource to comprehend the molecular events occurring during such a tightly regulated process. Using cDNA microarray, expression profiling of GC-1 and GC-2 cell lines was done to precisely understand their characteristics and uniqueness. Our observations indicate that whilst both the cell lines are indeed of testicular origin, GC-2 is not haploid as was originally thought. Data analysis of the 23,351 transcripts detected in GC-1 and 20,992 in GC-2 cell lines demonstrates an 80% transcript overlap between GC-1 and GC-2 cells and ~ 40% similarity of both with the primary spermatocyte transcriptome. 3152 and 793 transcripts exclusive to GC-1 and GC-2, respectively, were identified. The presence of transcripts for 36 genes was validated in these cell lines including those showing testis-specific expression, as well as genes not reported previously. Overall, this study provides the transcriptome database of GC-1 and GC-2 cells. Analysis of the data demonstrates the transcriptomic transitions between GC-1 and GC-2 thus providing a glimpse to the process of germ cell differentiation from type B spermatogonium into preleptotene spermatocyte.

Spermatogenesis is an intricate biological event wherein an undifferentiated spermatogonium develops into mature sperms. MicroRNAs are a type of single strand small non-coding RNA molecule and are implicated in the regulation of many crucial pathways during cell proliferation, apoptosis, and differentiation.Here, we present a comprehensive comparison of miRNA expression profiling in three main stages during porcine spermatogenesis using high-throughput sequencing.We built three small RNA libraries for the testis, the epididymis and the ejaculated sperm from a Landrace boar, and in total obtained 3821 precursor hairpins encoding for 4761 mature miRNAs, of which 23 are miRNA*. Notably, 940 precursor miRNAs produced both the 5'- and 3'- strands as sister pairs, indicating the distinctive expression patterns of germ cell miRNAs. Additionally, 418 out of 710 co-expressed miRNAs were identified as being differentially expressed between libraries (P 0.001). Apart from the sexual specific X chromosome, many miRNAs were found to be located on chromosome 12, which may play potential roles in spermatogenesis according to the result of synteny analysis with human and mouse. The Gene Ontology and KEGG pathway analysis revealed that the target genes of co-expressed miRNAs were highly involved in the cell cycle process, metal ion binding, modification of plasma membrane, and the p53 signal pathway.

The overpopulation of abandoned and stray companion animals has become a global crisis. The main purpose of this study was to develop a novel nanomedicine-based antifertility compound for non-surgical castration of male animals. Mangosteen (Garcinia mangostana L) pericarp extract has been shown to exhibit anti-fertility property. α-mangostin (AM)-loaded nanostructured lipid carrier (AM-NLC) was developed to improve male germ cell apoptosis. This study was conducted to investigate physicochemical properties of AM-NLC and determine the biological effects of AM-NLC on spermatogonia cells and testicular explants obtained from castrated testes. AM-NLC was produced through a hot homogenization technique. The negatively charged particle of AM-NLC was nano-sized with a narrow dispersity. AM-NLC exhibited antiproliferative activity towards spermatogonium cells. It induced apoptosis in the cells. In addition, AM-NLC exhibited anti-inflammatory activities in lipopolysaccharide-activated macrophages. Abnormal anatomy of seminiferous tubule was noted following treatment of testicular explant with AM-NLC. This nanomedicine-based sterilant would be a promising platform that may have utility in non-surgical castration of male animals by intra-testicular injection.

Androgens mediate a wide range of physiological responses and developmental processes in vertebrates, involving both reproductive and nonreproductive systems. The activity of androgens is mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily. In this study, an AR gene was cloned from the large yellow croaker (Larimichthys crocea) for the first time. qRT-PCR revealed ubiquitous expression of AR in all adult tissues examined, with higher expression in the gonad and liver of both sexes and highest expression in the blastula stage of embryonic development. Using in situ hybridization, we detected positive signals of AR in the spermatogonium, spermatocyte, spermatid, and spermatozoon during spermatogenesis, in the cytoplasm of all oocytes during oogenesis and in the follicle cells of stage IV oocytes. Our findings support the important role that AR plays in gametogenesis, gonadal development, and the early stages of embryonic development.

Spermiogenesis is a developmental process undergoing continuous differentiation to drive a diploid spermatogonium towards a haploid sperm cell. This striking transformation from spermatogonium to spermatozoa is made possible by the stage-specific adaption of cytoskeleton and associated molecular motor proteins. KIFC1 is a C-terminal kinesin motor found to boast essential roles in acrosome biogenesis and nuclear reshaping during spermiogenesis in rat. To explore its functions during the same process in Macrobrachium nipponense, we have cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed mn-KIFC1) from the total RNA of the testis. The 2,296 bp mn-KIFC1 cDNA contained a 87 bp 5' untranslated region, a 211 bp 3' untranslated region and a 1,998 bp open reading frame. Protein alignment demonstrated that mn-KIFC1 had 37.7, 58.7, 38.4, 37.2, 38.9 and 37.8% identity with its homologues in Salmo salar, Eriocheir sinensis, Homo sapiens, Mus musculus, Danio rerio and Xenopus laevis respectively. The phylogenetic tree revealed that mn-KIFC1 is most related to E. Sinensis KIFC1 among the examined species. Tissue expression analysis showed the presence of mn-KIFC1 in the testis, hepatopancreas, gill, muscle and heart. In situ hybridization showed that the mn-KIFC1 mRNA was localized at the periphery of the nuclear membrane and in the proacrosomal vesicle in early and middle spermatids. In late spermatids and spermatozoa, mn-KIFC1 was expressed in the acrosome and in the spike. In situ hybridization also indicated that KIFC1 works together with lamellar complex (LCx) and acroframosome (AFS) to drive acrosome formation and cellular transformation. LCx and AFS have both been previously proved to have essential roles during spermiogenesis in M. nipponense. In conclusion, the expression of mn-kifc1 at specific stages of spermiogenesis suggests a role in cellular transformations in M. nipponense.

Spermatogenesis and spermiogenesis of the monozoic caryophyllidean cestode Wenyonia virilis (Woodland, 1923), an intestinal parasite of Synodontis schall (Pisces: Siluridae), have been investigated by means of transmission electron microscopy for the first time in Egypt. Spermatogenesis is of a rosette type; each spermatogonium undergoes four mitotic and two meiotic divisions resulting in the formation of sixty four spermatids. These spermatids undergo spermiogenesis and transform into mature spermatozoa. The process of spermiogenesis begins by the formation of the zone of differentiation, at one end of the spermatid cell, with one pair of centriole but without an intercentriolar body. The left-handed centriole, firstly, forms a basal body but aborts forming a flagellar bud. The right-handed one develops externally into an axoneme growing parallel to a cytoplasmic extension protruding from the differentiating zone. The nucleus penetrates this extension followed by the proximodistal fusion of the axoneme in the sperm shaft. The spermatozoon of W. virilis lacks mitochondria and consists of five regions showing gradual increase in the nucleus width and the presence of glycogen granules. Some important differences between the present study and others done on monozoic and polyzoic worms are recorded and discussed.

The gonad of Spratelloides gracilis was not sexually differentiated in the yolk-sac, preflexion, flexion and postflexion stages. Sexual differentiation and development of the ovary and testis started in the transition stage from larva to juvenile. In juveniles at the fin ray completion stage, the ovary and testis could be distinguished because the ovary contained germ cells initiating meiosis and the testis had blood vessels and a high density of somatic cells. The ovary further developed in larger juveniles to have oocytes of perinucle olus stage together with those of the chromatin nucleolus stage, and oogonium. However, in the testis of larger juveniles, primary spermatogonium began prolife ration by meiosis. Sexual differentiation may be regarded as one of morphological and functional changes accompanying metamorphosis in S. gracilis. Some fish larger than the mature size of 60mm standard length had advanced germ cells and functional gonads, others did not have functional gonads. The distal end of the immature gonads did not connect with a genital duct near the anus. These observations indicate that S. gracilis has large variability in size-at-maturity. The variability in size-at-maturity in S. gracilis, together with large variability in age-at-maturity, may constitute an ecological basis for an extended spawning season in S. gracilis.

The light microscopic structure of the testis and genital duct system of the freshwater stingray Himantura signifer was observed. The testis is composed of lobes having numerous spermatocysts in a dorsoventral zonated arrangement. The germinal papilla at the middorsal surface of the testicular lobe is the origin site of spermatocyst development, where mesenchymal-like cells are predominantly found. The association of a Sertoli cell precursor with a spermatogonium marks the onset of spermatocyst formation and development. The newly formed spermatocysts at the dorsal end of the germinal zone replace the older ones, which are sequentially moved to the ventral side and are termed spermatogonial, spermatocyte, spermatid, spermatozoal, and degenerate zones. In the degenerate zone, the spermatocysts deteriorate after releasing the spermatozoa into the intratesticular duct, where they are further transported through the extratesticular duct system and finally stored at the seminal vesicle. The epithelial lining of the genital duct is a pseudostratified ciliated columnar with no muscular layer underneath; thus, sperm are conveyed through ciliary activity. The interesting features of the present study are the finding of mesenchymal-like cells in the germinal papilla and the nonaggregated formation of sperm in the seminal vesicle.

PubMedID: 14618398 Ultrastructure of the membrana propria and the seminiferous epithelium was studied in infertile human testis both before and 3-6 months after varicocelectomy. The frequent alterations, observed before and after the operation, were extremely thickened membrana propria, deep invaginations, multilamination and knob-like formation of basal laminae and formation of multinucleated spermatids, which were all considered as the common response of the testis to different noxious agents. Although the cells of the seminiferous epithelium were clearly affected by varicocele before varicocelectomy, many areas exhibited normal features after the operation. Furthermore, multinucleated cells, sharing common features of Sertoli cell and spermatogonium, were observed, as well as presence of well-developed annulate lamellae in the Sertoli cells, exhibiting centrioles in the vicinity of their nuclei after varicocelectomy. These multiple ultrastructural observations indicate that Sertoli cell division takes place. This study suggests that if the observation period of the tissue samples after varicocelectomy is long enough, the reversible changes of the tubular cells would be seen much more frequently.

We used immunolocalization in tissue sections and cytogenetic preparations of female and male gonads to study the distribution of the proliferation marker pKi-67 during meiotic cell cycles of the house mouse, Mus musculus. During male meiosis, pKi-67 was continuously present in nuclei of all stages from the spermatogonium through spermatocytes I and II up to the earliest spermatid stage (early round spermatids) when it appeared to fade out. It was not detected in later spermatid stages or sperm. During female meiosis, pKi-67 was present in prophase I oocytes of fetal ovaries. It was absent in oocytes from newborn mice and most oocytes of primordial follicles from adults. The Ki-67 protein reappeared in oocytes of growing follicles and was continuously present up to metaphase II. Thus, pKi-67 was present in all stages of cell growth and cell division while it was absent from resting oocytes and during the main stages of spermiocytogenesis. Progression through the meiotic cell cycle was associated with extensive intranuclear relocation of pKi-67. In the zygotene and pachytene stages, most of the pKi-67 colocalized with centromeric (centric and pericentric) heterochromatin and adjacent nucleoli; the heterochromatic XY body in male pachytene, however, was free of pKi-67. At early diplotene, pKi-67 was mainly associated with nucleoli. At late diplotene, diakinesis, metaphase I and metaphase II of meiosis, pKi-67 preferentially bound to the perichromosomal layer and was almost absent from the heterochromatic centromeric regions of the chromosomes. After the second division of male meiosis, the protein reappeared at the centromeric heterochromatin and an adjacent region in the earliest spermatid stage and then faded out. The general patterns of pKi-67 distribution were comparable to those in mitotic cell cycles. With respect to the timing, it is interesting to note that relocation from the nucleolus to the perichromosomal layer takes place at the G2/M-phase transition in the mitotic cell cycle but at late diplotene of prophase I in meiosis, suggesting physiological similarity of these stages.

The spermatogenesis of Allostoma sp. and the organization of mature spermatozoa are described by means of transmission electron microscopy. All stages of sperm development were observed in mature testes, which are bordered by a layer of fibrous connective tissue. The primary spermatogonium produces 64 spermatids, grouped as a cytophore. The process of spermiogenesis occurs at the periphery of the cytophore, when the sperm body is formed and released at complete maturation. The mature spermatozoon is a flattened, filiform structure. It is aciliary. Its anterior end is not distinguishable from the posterior one in the testis. The nucleus, showing an approximately rectangular cross section, is situated centrally and the chromatin is densely packed. A membranous cylinder, located on each side of the nucleus, contains conspicuous stacks of clear thick-envelope vesicles, and a long mitochondrial derivative placed peripherally. The vesicles show contacts with the cylinder membrane and the outer sperm membrane. Their supposed Golgi-derived content may thus be released into the external medium. A hypothesis is proposed about the role of these vesicles in sperm motility.

The stages of the cycle of the seminiferous epithelium in the Japanese macaque are investigated using testes fixed by a mixture of formaldehyde and glutaraldehyde containing picric acid and embedded in a methacrylate resin, Quetol 523M. Sections, 1.0–2.0 µm in thickness, were cut with glass knives and stained with periodic acid-Schiff (PAS) and hematoxylin. Sections from such resin blocks illustrated cellular detail without structural distortion during the polymerization process. Furthermore, staining affinity with PAS and hematoxylin was excellent. In stained sections, typical germ cell associations were described, based on the nuclear morphology of type A (dark and pale) spermatogonium, type B spermatogonium, various developmental stages of primary spermatocytes during meiosis, and the development of the acrosomic system. In the Japanese macaque, two different steps of spermatids (steps 3 and 4) were constantly seen in the same area of the tubular epithelium during stage III. Therefore, a classification into ten stages is proposed for the cycle in this species. Additional characteristics are described based on the observation of the seminiferous epithelium using semithin sections.

The spermatogenesis and spermatozoon of Dicrocoelium dendriticum were studied by transmission electron microscopy. Peripheric accessory cells project between germ cells. Each spermatogonium gives rise to 32 spermatozoa. The stages in spermiogenesis include development of the zone of differentiation, appearance of the intercentriolar body flanked by two centrioles from each of which a free axoneme and a striated rootlet grow, outgrowth of the differentiation zone to form the median cytoplasmic process and migration of the nucleus and mitochondria into it, and rotation of the flagella and subsequent proximodistal fusion of the three projections to form a monopartite spermatozoon. The spermatozoon possess two incorporated axonemes with the "9 + 1" pattern typical of those in trepaxonematid plathelminths. beta-Glycogen particles accumulate in the spermatozoa after they have separated from the cytophore as revealed by Thiery's method. This study confirms in a further family, Dicrocoeliidae, the constant pattern of spermiogenesis and spermatozoon structure in Digenea.

The fine structure of the spermatogonium, spermatocyte and spermatid of a marine snail, Littorina sitkana is described. The ring centriole (annulus) is formed from the distal centriole and it migrates to the base of the mitochondrial region where it lies in a joint-like structure which is formed by an area of invaginated plasma membrane. The distal and proximal centrioles are at first perpendicular to each other but the proximal centriole rotates to a position coaxial with the distal centriole and fuses with it. The peripheral doublet fibers are continuous between the two centrioles but the central fibers originate only in the distal centriole. The acrosome differentiates from the proacrosomal granule which is derived from a Golgi body. Microtubules, present at this stage, may assist acrosomal formation. Chromatin condensation begins with the formation of fibrous strands, then to lamellar plates which become folded and later twisted around the flagellar shaft. In the final stages the lamellae appear in cross section as concentric rings which eventually fuse to form a homogeneously dense nuclear tube.

The cycle of the seminiferous epithelium of the nine-banded armadillo can be divided into ten stages. As in most mammals, only one stage is observed per tubular cross-section. The process of spermiogenesis can be divided into thirteen steps according to the development of the acrosomal system and the flagellum. Four generations of spermatogonia are observed in the germinal epithelium: 1) stem cells, 2) type “A”, 3) intermediate, and 4) type “B” spermatogonia. The stem cell is characterized by a highly irregular nucleus and the presence of glycogen in its cytoplasm. The type “A” spermatogonium contains an oblong nucleus with one or two shallow infoldings of the nuclear membrane. The intermediate spermatogonium contains an ovoid nucleus characterized by one or two nuclei and heterochromatin scattered in the nucleoplasm. The nucleus of the type “B” spermatogonium is more spherically shaped with a centrally placed nucleolus and heterochromatin associated with the nuclear envelope.

The DNA content was studied histographically in the nuclei of the cells of certain mitorically dividing tissues (spermatogonium, fibroblasts of albino mice, liver cells and those of the pancreas in young albino rats). The authors came to a conclusion, that the process of DNA reduplication is nonuniform. Each subsequent stage of the DNA synthesis is more intense than the preceeding one; the rate of synthesis increases during the second half of the process, reaching the maximum near the end of reduplication.

A light microscopic and phase-contrast study of the developing seminiferous follicles in two elasmobranch species was made. After an initial period of independent mitotic divisions, each spermatogonium becomes entirely surrounded by a cytoplasmic extension of a single follicle cell (Sertoli cell). These bicellular units may be termed spermatocysts. This relationship remains through six succeeding germ cell generations, usually resulting in a group of 64 isogenous spermatids in each spermatocyst. Thus, all substances exchanged between the blood stream and the germ cells must pass through the body of the follicle cell. As the spermatids transform into spermatozoa, they are drawn into a compact bundle and are finally released from the follicle cell. Germ cell development is closely synchronous within each spermatocyst and spermatocyst development is synchronous within each follicle. Germ cell and Sertoli cell lines appear to be entirely separate. Intercellular cytoplasmic connections are retained among large groups of daughter germ cells. It is suggested that these cytoplasmic bridges as well as spermatocyst formation serve in promoting equality of physiological opportunity throughout the large germ-cell mass.

A simplified method of organ culture of human gonads is suggested. Altogether 190 gonads from 7–11-week embryos were grown on Millipore filters in penicillin flasks. During the first week of culture areas of central necrosis were observed in both the ovaries and the testes, but during subsequent cultivation these disappeared in some of the explants. Progressive development of germinative cells took place in the ovaries of 9–11-week embryos as far as the pachytene stage, whereas development in the ovaries of 7–9 week embryos did not reach the leptotene stage. Germinative cells during cultivation of the testes did not progress beyond the spermatogonium stage.

The fine structure of Sertoli cells in three marine prosobranch molluscs has been studied with light- and electron microscopy. Sertoli cells of prosobranchs are modified columnar epithelial cells that maintain continuous contact with the basal lamina and extend from it to the lumen of a testicular tubule. Spermatogenesis takes place between adjacent Sertoli cells, but a continuous layer of cytoplasm separates the spermatogonia from the basal lamina, thus restricting the basal compartment to spermatogonium mother cells. Substances traversing the basal lamina from the interstitial space must pass either through or between the Sertoli cells. However, between the cells, a permeability barrier composed of septate and desmosome-like junctions blocks the passage of substances, such as the tracer lanthanum nitrate. The basally-located nucleus is irregularly shaped with fine granular euchromatin and some peripheral heterochromatin: satellite karyosomes border the nucleolus. There is an extensive intracellular digestive system that is used effectively to phagocytize waste sperm and residual cytoplasm. Cytoplasmic processes of Sertoli cells penetrate throughout the germinal epithelium. In some prosobranchs that exhibit sperm polymorphism these processes must coordinate to bring together a clone of eupyrene sperm and a carrier sperm at a particular time in development. The only cytoskeletal elements available within the processes to generate such movements are microtubules. We propose that the term 'nurse cell', which has been used in the past to describe at least three different cell types, including Sertoli cells and apyrene sperm, be restricted to abortive oogonia that contribute to development of an oocyte.

IT has been known for many years that Carnoy, Gilson, Petrunkevitch, corrosive acetic, Bouin, and a host of ordinary fixatives will enable remains of mitochondria to be demonstrated with staining in alum haematoxylin or acid fuschsin. This applies especially to cytoplasmic inclusions when aggregated as in the spermatogonium, or in the mitochondrial nebenkerne. Mitochondria often show nicely after acetic acid containing fixatives such as the ‘Susa’ mixture or Flemming with acetic acid. This applies especially to all vertebrate tissues. With most invertebrate tissues such methods are deadly, and if mitochondria remain they are almost certainly badly distorted. Practically all the beautiful work of Meves was done with acetic acid containing fixatives. This is why he left so much to be done by those who followed.

WITH reference to Dr. R. Sparck's statement (NATURE, October 7, p. 480) that the male stage in the oyster is due to the coldness of the temperature, it should be pointed out that in various hermaphrodite mollusca, such as Helix and Arion, the reason for the passage of the indifferent epithelial cell, either to oogonium or spermatogonium, is at present unknown. Older authors considered that those cells near yolk, or near a superior nutrimental radius became eggs, and that those less exposed to steady streams of nourishment became spermatocytes.