Differential Activity of Stanniocalcin in Male and Female Fresh Water Teleost Mastacembelus armatus (Lacepede) during Gonadal Maturation

The present study was carried out to analyze the differences in the activity of hormone stanniocalcin (STC) between male and female fishes of Mastacembelus armatus during their gonadal cycle. A large variation in nuclear diameter of cells of corpuscles of Stannius (CS) were recorded in relation to testicular cycle as well as ovarian cycle which indicates that the cellular activity varied with different phases of reproductive cycle in both male and female fish. Similar changes in nuclear diameter of CS cells were also observed after 17alpha-methyltestosterone administration in males and 17 β-estradiol administrations in females. A positive correlation was observed between plasma STC levels, gonadosomatic index (GSI) and the sex steroids in both sexes, suggesting that STC has a role in the processes involved in gonadal development. In addition females showed remarkable changes in plasma calcium level during gonadal cycle while no such change for males were observed. In females the plasma calcium level estimated during different phases of reproductive cycle indicates positive correlation between plasma level of calcium and gonad growth. Thus hyperactivity of CS cells was noted in both male and female fishes during gonadal cycle along with the differences in the activity of STC as well. In female it may act as hypocalcemic factor and bring the level of calcium to normal which increases during preparatory and pre spawning phases to fulfill the increased demand of calcium for vitellogenesis. However data of male fishes indicated that plasma STC concentration varied widely during gonadal cycle but showed no consistent relationship to plasma calcium level.     

Studies of gonadal sex differentiation

Gonadal differentiation has a determinative influence on sex development in human embryos. Disorders of sexual development (DSD) have been associated with persistent embryonal differentiation stages. Between 1998 and 2015, 139 female patients with various (DSD) underwent operations at the Scientific Center of Obstetrics, Gynaecology and Perynatology in Moscow, Russia. Clinical investigations included karyotyping, ultrasound imaging, hormonal measurement and investigations of gonadal morphology. The male characteristics in the embryo are imposed by testicular hormones. When these are absent or inactive, the fetus may be arrested at between developmental stages, or stay on indifferent stage and become phenotypically female. A systematic analysis of gonadal morphology in DSD patients and a literature review revealed some controversies and led us to formulate a new hypothesis about sex differentiation. Proliferation of the mesonephric system (tubules and corpuscles) in the gonads stimulates the masculinization of gonads to testis. Sustentacular Sertoli cells of the testes are derived from mesonephric excretory tubules, while interstitial Leydig cells are derived from the original mesenchyme of the mesonephros. According of the new hypothesis, the original mesonephric cells (tubules and corpuscles) potentially persist in the ovarian parenchyma. In female gonads, some mesonephric excretory tubules regress and lose the tubular structure, but form ovarian theca interna and externa, becoming analogous to the sustentacular Sertoli cells in the testis. The ovarian interstitial Leydig cells are derived from intertubal mesenchyme of the mesonephros, similar to what occurs in male gonads (testis). Surprisingly, the leading determinative factor in sexual differentiation of the gonads is the mesonephros, represented by the embryonic urinary system.     

On the cytoarchitecture of the corpuscles of Stannius of the catfish, Heteropneustes fossils (Bloch).

The cytoarchitecture of the corpuscles of Stannius (CS) of the catfish, Heteropneustes fossilis has been investigated. Based on the arrangement of septa and cells, at least 4 principal architectural patterns can be distinguished. In the first type, the cells appear either as cords or as follicles due to complete formation of thin septa. In the second category also, the penetrating septa are thin but divide the CS into incompletely delimited lobes. In the third type, the septa are thick and their branches unite, sometimes resulting in the formation of complete lobes. In the fourth type, each CS is formed of aggregates of lobes, each of which consists of a number of complete or incomplete lobules. The limitations involved in the classification of the CS into 4 types, on the basis of the septal arrangement, have been discussed in the light of the fact that H. fossilis possesses all the 4 types of CS.     

Cytological changes in the pars distalis of pituitary of the green frog,Rana esculenta L., during the reproductive cycle

Summary The cytological changes in the pars distalis of pituitary of male and female green frog, Rana esculenta during the reproductive cycle have been studied by examining the form, cytoplasmic granulation and numerical proportions of the different cell types. Some sex differences were recorded, particularly in relative total number of cells in a median sagittal section of the pars distalis. No extensive cytological changes occur during the hibernation period, but there are striking alterations at the time of spring awakening, during spawning months and during replenishment of the spent gonads i.e. during late summer and autumn months. Important changes are to be observed in the acidophils type one (A1) and basophils type two (B2) and three (B3). Changes in the A1 cells during breeding season have been speculated as being in support of the spawning activity of the animal. The B2 cells exhibit most profound changes which correlate precisely with the morphological changes in gonads, and these cells are considered as the FSH-gonadotrops. The functional significance of the B3 cells is discussed and it is suggested that they might be the source of an ICSH- or LH-like hormone.The work has been supported by grant from the National Research Council of Italy.The award of postdoctoral fellowship by the National Research Council of Italy is gratefully acknowledged.     

Stannius corpuscles and plasma calcium levels during the reproductive cycle in the cichlid teleost fish,Oreochromis mossambicus

Summary The corpuscles of Stannius (CS) of the cichlid Oreochromis mossambicus (formerly Sarotherodon mossambicus) were studied in relation to sexual maturation and plasma calcium levels. After sexual maturation, the CS are enlarged in female fish, because of an increase in size and number of the type-1 cells. During the ovarian cycle, the size of the CS increases in parallel with the growth of the ovaries. Concurrently, plasma total calcium increases markedly until spawning. This increase is mainly accounted for by calcium bound to proteins (vitellogenins), but the ultrafiltrable calcium fraction is also slightly higher than in males. Ovariectomy is followed by a reduction in the size of the CS, mainly a result of involution of the type-1 cells, and by a reduction in plasma calcium to levels typical for males. Gonadectomy in males does not affect size or ultrastructure of the CS, or plasma calcium levels. Since the type-1 cells of the CS are the presumptive source of a hypocalcemic hormone, we conclude that activation of the CS during the female reproductive cycle is a response to elevated calcium levels that accompany ovarian maturation. We suggest that the CS respond in particular to the elevated ultrafiltrable or ionic calcium levels.     

Synthesis of glycoconjugates in mouse primordial germ cells

The synthesis of protein-bound carbohydrates has been studied in primordial germ cells (PGCs) and in somatic cells of 12.5 to 13.5-days-postcoitum (dpc) fetal mouse gonads. Both cell types were shown to synthesize asparagine-linked glycopeptides and glycosaminoglycans (GAGs). In addition, PGCs also synthesize lactosaminoglycans (LAGs) although in different proportions in female and male germ cells. Female PGCs, which at 13.5 dpc are entering meiosis, synthesize mainly LAGs, and minor amounts of hyaluronic acid (HA) and chondroitin sulfate (CS). Male germ cells, on the other hand, synthesize mainly CS. Furthermore, somatic cells of fetal gonads synthesize HA as the major class of GAGs. It is suggested that the activation of LAG synthesis in developing germ cells might be related to the beginning of meiosis. Moreover, we propose that HA synthesis might be developmentally regulated in somatic cells of the gonad, in order to regulate the establishment of specific interactions with germ cells.     

Embryonic origin and development of the corpuscles of Stannius in chum salmon (Oncorhynchus keta)

Summary An immunocytochemical technique was used to follow the embryological origin and development of the corpuscles of Stannius (CS) in the chum salmon, Oncorhynchus keta. Stanniocalcin immunoreactive (ir-) cells can be observed as early as 13 days before hatching. The ir-CS cells appear in clusters of variable size in close association with nephric ducts. In addition, individual ir-cells also occur at this stage amoung epithelial cells of the nephric ducts. these individual cells may give rise to clusters which subsequently increase in size, the largest reaching 100 m in diameter by the time of hatching. During this period, dispersed CS cells become evident and develop into secondary clusters in the vicinity of the primary clusters. These clusters appear to fuse to form larger clusters with a lobular structure. Transfer of the larvae (20 days after hatching) from fresh water to 50% seawater, accelerates the development of the CS tissue, suggesting an important role of the CS in seawater adaptation.     

Cytochemistry of histones throughout the division cycle in meristematic cells

Summary We have been able to follow the increase in nuclear histones along the division cycle, using a natural synchronous meristematic cell population labelled by caffeine as binucleate. We have thus found a parallel increase in DNA and histone along the S period. Some increase in nuclear histones during the G₁ and G₂ has also been detected. At the same time a high rise of stainable histone in the nuclei in prophase has been found.Changes in the composition of nuclear histones have been followed counting the percentage of arginine-rich histones in the total histone content. Two peaks located at the beginning and at the end of the S period as well as a sharp descent of the percentage of arginine-rich histone in the prophasic nucleus have been found.     

Are Stannius corpuscles the parathyroid glands of teleost fishes? Ultrastructural, cytologic and immunocytochemical arguments

Parathyrin of Stannius corpuscles (PCS), glands which are restricted to Holostei and Teleostei, is closely related to mammalian parathyrin (PTH) secreted by the parathyroids [( 6] to [9]). In unstimulated and stimulated CS we have shown the same structure and cellular types that are described in mammalian parathyroids. We observed three types of cells; the two sorts of cells already described [14] present such a difference of density (Fig. 1) that type I may be compared to chief dark cells and type II to chief light cells. The difference between these two forms was particularly marked in activated CS; a similar observation has been reported concerning PT [15]. Furthermore, we have detected a third type of cell present either singly in unactivated CS or in small groups between chief cells in activated CS. They showed all the characteristics of oxyphil cells [17]; they present an extremely dense cytoplasm with numerous mitochondria and a typical stellate form with cytoplasmic processes extending between chief cells (Figs. 2, 3). In CS of untreated eels, we have shown by means of indirect immunocytology (using an immunserum raised against the active fragment 1-34 bPTH) that the immunostained reaction product was limited to dilated cisternae and ribosomes of the rough endoplasmic reticulum and to most of the granules in all the chief cells (Figs. 4, 5). No immunoreaction was observed in Golgi area. Oxyphil cells did not present an immune localization of PCS. CS are structurally and cytologically similar to mammalian PT; furthermore their chief cells synthesize, stock and secrete a substance immunologically similar to mammalian PTH; the exact function of oxyphil cells has to be demonstrated.     

Age-related changes in the serotonin content of the epiphysis of birds

Serotonin content of the epiphysis has been studied in 1-day chicks, 1-, 5-5 1/2-, 8-12- and 24-month hens. It was found that this content undergoes significant changes during life cycle of hens, being dependent on physiological activity of the gonads. The highest level of serotonin (16 micrograms/g) was observed in 8-12 months old hens which corresponds to the period of the most intensive activity of the gonads.     

Synchronization of porcine fetal fibroblast cells with topoisomerase-inhibitor hoechst 33342

Proper synchronization of donor nuclei has been shown to have a major influence on the developmental potential of nuclear transfer embryos. In the present study, a protocol was established to synchronize porcine fetal fibroblasts in the G2 stage of the cell cycle. Cell cycle analyses were performed by flow cytometry. Cells were pre-synchronized by serum deprivation or aphidicoline-treatment; then incubated in medium containing 0.1 microg/ml Hoechst 33342 (H342). The fluorochrome H342 has been shown to be a topoisomerase-inhibitor that can inhibit progression through the cell cycle. There was no significant difference in the percentage of fibroblasts in G2/M whether cells were pre-synchronized in medium supplemented with 0.1% serum for 48h or 0.5% serum for 6 days. Compared with controls, pre-synchronization in early S-phase before incubation in H342 increased the proportion of G2/M fibroblasts; also an increase from 0 and 6 versus 12h culture in complete medium before incubation in H342 resulted in an increased percentage of cells in G2/M at the end of the synchronization period (9.3 and 13.1% versus 33.7%; P<0.001). Neither an increase in the concentration of H342 (0.1-1.0 microg/ml) nor a longer exposure time (12h versus 18h versus 24h) increased the proportion of G2/M fibroblasts. The protocol established in this study arrested porcine fibroblasts reversibly in the G2/M-stage and is therefore suitable to provide synchronized cells for nuclear transfer experiments.     

Differential localization of vasopressin- and oxytocin-immunoreactive cells and conventional neurosecretory cells in the ganglia of the earthworm Pheretima hilgendorfi

Cells containing arginine vasopressin (AVP)- and oxytocin (OXT)-like substances were immunohistochemically visualized in the cerebral, subesophageal, and ventral nerve cord ganglia of the earthworm Pheretima hilgendorfi. Whether these anti-AVP– and anti-OXT–reactive cells are identical with classical aldehyde fuchsin (AF)-positive neurosecretory cells was tested in serial sections. In all ganglia, groups of scattered neuronal cell bodies and axons strongly reactive to AVP and OXT antisera were observed, but AF-positive cells consisting of type a (dark blue) and type b (purple) cells were predominantly present in the cerebral and subesophageal ganglia. In the cerebral and subesophageal ganglia anti-AVP– and anti-OXT–reactive cells were generally larger than AF-positive cells. Some AF-positive cells were reactive either to anti-AVP or anti-OXT serum, but some failed to react to either serum. Anti-AVP– and anti-OXT–reactive cells were not immunoreactive to OXT and AVP antisera, respectively. Electron microscopic observations showed that the granules of type a cells were larger and less electron dense than those of type b cells and anti-AVP–reactive cells. The present cytological observations clearly showed that AVP- and OXT-like substances were widely present in the ganglionic cells of the earthworm     

Study of the testis in the active and hibernating hedgehog

The structure and ultrastructure of the hedgehog's testis has been studied during active and hibernating states. The maximum activity of the hedgehog's testis manifest itself in spring and summer. In autumn they enter in a seasonal resting, in which, the interstitial cells atrophy and it can observe primary spermatocytes resting next to spermatogonia and supporting cells in the seminiferous tubules. These have a minimal diameter and contain only primary spermatocytes in hibernating period also the seminiferous epithelium that compound the walls of the tubules are about 72 microns thick in the active period and about 47 microns in hibernation. The Sertoli cells shows a small diameter in hibernation than in the active season. The nucleus has one or two nucleoli with distinguish themselves from those observed in the active season because of their closer union with the inner nuclear envelope. In autumn (resting period) the interstitial tissue regress and the Leydig cells are aggregated in blocks with little cytoplasm, minimum size nuclei and 1 or 2 nucleolus closely to the inner nuclear membrane. Like then in the ovary, the hedgehog's testis follows a seasonal cycle determined by the temperature and environmental changes and it have a power of physiological adaptation inherent of a typical hibernating animal.     

Cell cycle analysis of cultured porcine mammary cells

One of the major points of debate in determining the effectiveness of nuclear transfer technology has been the phase of the cell cycle of the donor cell at the time of nuclear transfer. Here, a primary mammary cell line has been isolated and various treatments for synchronization of the cell cycle have been tested. The cells were then simultaneously stained for DNA content and protein content and the percentages of cells in G1, G0, S, and G2 + M were estimated. In the first experiment, cells were either cycling, grown to confluence, or serum-starved for 5 days. Serum starvation increased (p < 0.05) the percentage of cells in G0 compared to confluent or cycling cells from 3% to 8% to 22%. By using forward scatter to determine the size of the cells it was determined that if small cells (7-15 microm) were selected from the serum-starved group 43.9% will be in G(0) as compared to 4.5% of cycling cells and 9.9% of confluent cells. Dimethyl sulfoxide (DMSO) treatment (0%, 0.5%, or 1.0%) for 72 hours (shown to synchronize some cell types in G0) had no effect on the percentage of cells in G0, G1, S, or G2 + M. Treatment with mimosine (0 microM, 0.4 microM, 0.8 microM or 1.2 microM), a compound that should synchronize the cells in G1, increased (p < 0.05) the percentage of cells in G1 from 66.7% (0 microM mimosine) to 79.0% to 82.0%. Finally, treatment with colchicine for 24 hours (shown to synchronize some cell types in G2 + M) increased (p < 0.05) the percentage of cells in G2 + M (0 microM colchicine) from 13.3% to 27.2% to 31.6%. It is concluded that many cell cycle synchronization techniques are effective in porcine mammary cell lines, but none of the techniques are 100% effective. Such results should help elucidate the mechanisms involved in nuclear transfer.     

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complex II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 ρ(0) cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in ρ(0) cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.     

Immunocytochemical localization of hypocalcin in the endocrine cells of the corpuscles of Stannius in three teleost species (trout,flounder and goldfish)

Summary In order to identify the cell-type responsible for the production of hypocalcin (the recently isolated hypocalcemic hormone of teleost fish), the corpuscles of Stannius (CS) of trout, flounder and goldfish, were immunocytochemically stained with antisera raised against trout hypocalcin. The secretory granules of the type-1 cells of the CS, considered to be the hypocalcin-producing cells, showed intense immunoreactivity in all species examined. However, in trout and flounder, the secretory granules produced by the type-2 cells, which have been suggested to represent a functionally different cell-type, also showed an intense immunoreactivity. In goldfish, no type-2 cells were observed. We tentatively conclude that type-1 and type-2 cells represent structurally different forms of the same functional cell-type.     

Electron microscopic studies of the corpuscles of Stannius of an airbreathing teleost (Heteropneustes fossilis)

The ultrastructure of the corpuscles of Stannius (CS) ofHeteropneustes fossilis reveals a homogenous cellular composition characterized by only one cell type, with large secretory granules and abundant ribosomal endoplasmic reticulum. These cells are comparable to the type 1 cell described in the CS of other teleosts; type 2 cells, whose presence is ubiquitous in the CS of freshwater species are absent inH. fossilis. Our data on the CS ofH. fossilis demonstrate that not all freshwater species possess type 2 cells in their CS and these are not essential for life in freshwater.     

Cell confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the cell cycle in granulosa and fibroblast cells of cattle

The cell cycle stage of donor cells is an important factor influencing developmental ability of nuclear transfer embryos. In the present experiment, cumulus and fibroblast cells of cattle were subjected to flow cytometric cell cycle analysis before being used in somatic cloning experiments. The following experimental groups were analyzed for each cell type: (1) actively dividing cells, (2) cells confluent for 4 days, (3) cells starved for 1, 2, 3 or 5 days. Using the propidium iodide flow cytometric assay, there were no significant differences (P > or = 0.05) in the percentage of cells in G0/G1 regardless of origin and type of cell, after confluency or serum starvation. Differences with the growing cells were found (P < or = 0.01). To determine what subset of cells in G0/G1 were in the G0 subphase of the cell cycle, an immunofluorescence analysis was conducted using monoclonal anti-PCNA antibodies in a FACS assay. There were not statistically significant differences in the percentage of cells that enter G0, between confluent and any starved group for either type of cells. Bovine fibroblast cells, confluent or serum starved for 3 days, were used in nuclear transfer experiments. A slight trend for a more desirable fusion rate in starved cells was detected, and embryo cleavage was greater in starved cells, however, in vitro development to blastocysts was similar between groups. Data indicate that prolonged culture of cells in the absence of serum does not imply a shift in the percentage of cells that enter G0/G1 or G0 alone, and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs, because there are negative effects such as apoptosis, associated with serum starvation.     

Annual changes in the ovarian activity of the freshwater teleost, Barbus luteus (Heckel) from Southern Iraq

The present paper describes the morphology, histology, and annual cyclical changes in the ovaries of Barbus luteus (Heckel). The ovaries are paired structures, joined by short oviducts. The ovarian structure consists of a number of lamellae, which contain the germ cells in various phases of maturation—immature, maturing and mature. Two types of yolk have been reported in the oocyte. The endocrine function of the gonads has been attributed to the follicular cells forming the 'pre-ovulatory corpus luteum' and 'postovulatory corpus luteum'. The ovaries undergo a regular annual cycle which has been divided into five stages, based on various criteria, viz, ovarian size, colour, gonosomatic index, average ova-diameter, histological composition, and the spawning activity.