Observations on rat sertoli ectoplasmic (‘junctional’) specializations in their association with germ cells of the rat testis

The ectoplasmic (‘junctional’) specialization, a subsurface modification of the Sertoli cell that is often seen facing germ cells, was studied in relation to the development and maturation of these germ cells. This structure is composed of sub-surface bundles of filaments and more deeply placed endoplasmic reticulum. The data indicate that these subsurface modifications of Sertoli cells are reutilized in a cyclic fashion, being transferred from their position facing late spermatids to one opposing less mature germ cells. Ectoplasmic specializations appeared to function mechanically in grasping the heads of the spermatids which are undergoing the elongation and maturation phases of spermiogenesis rather than in actually attaching Sertoli cells to these germ cells. It is postulated that the ectoplasmic specialization imparts rigidity to that area of the Sertoli cell that surrounds the head region of the germ cell, forming a recess and a mantle by which the germ cell may be moved toward the base or toward the surface of the seminiferous epithelium. The observed linkage of microtubules to the cisternae of the complex provided a morphological basis for the changes in the cytoarchitecture of the Sertoli cell, which must accompany these movements.     

A study of Sertoli-spermatid tubulobulbar complexes in selected mammals

Tubulobulbar complexes (TBCs) were found in nine mammalian species (opossum, vole, guinea-pig, mouse, hamster, rabbit, dog, monkey and human) primarily originating from the plasma membrane overlying the acrosome of late spermatids. Fewer complexes (4–10) were noted in these species than has been previously reported for the rat (up to 24). TBCs were not seen emanating from round spermatids or those elongated spermatids located within the deep recesses of the Sertoli cell, but they appeared as the spermatids came to reside much closer to the tubular lumen in preparation for release. TBCs developed in areas deficient or lacking in Sertoli filaments and endoplasmic reticulum (ectoplasmic specialization). In general their structural configuration was similar to that shown in the rat, although minor differences were noted. Fine fibrils were observed connecting the distal portion of the spermatid tube with the Sertoli plasma membrane forming a bristle-coated pit. The length of TBCs from most species studied was 1–2 μm, whereas those of the opossum extended 6–8 μm into an apical Sertoli process. TBCs were degraded within the Sertoli cell by its lysosomes prior to sperm release, and for most species there was evidence indicating that formation of more than one generation of TBCs occurred. As sperm release approached, TBCs formed preferentially from the leading edge of spermatids with spatulate heads. The Sertoli cell gradually withdrew from around the spermatid head until only the tip of the head was embedded within the Sertoli cell. This region of contact frequently demonstrated TBCs. The proposed functions of TBCs are reviewed and discussed in light of these findings from other species.     

Increased serum prolactin levels mediate the suppressive effects of ectopic pituitary grafts on copulatory behavior in male rats

To determine if deficits in sexual activity observed in pituitary-grafted male rats are due to elevated serum prolactin (PRL) levels found in these animals, the effects of whole pituitary grafts, pars distalis grafts, and ovine (o) PRL treatment on male copulatory behavior were compared. Adult sexually experienced CDF male rats were given four whole pituitary grafts, four pars distalis grafts, or were sham operated. Both groups of grafted animals exhibited suppressed copulatory behavior patterns when tested 18 days after pituitary transplantation. Animals given whole pituitary grafts had significantly longer latencies to mount (P less than 0.05) and to intromit (P less than 0.01) than did the sham-operated controls, while the animals given anterior pituitary grafts differed from the sham-operated controls in latencies to mount (P less than 0.05) and to intromit (P less than 0.01), as well as in the number of intromissions (P less than 0.05). Prolactin-injected animals had significantly reduced intromission rates (P less than 0.01) and significantly increased latencies to mount (P less than 0.05) and to intromit (P less than 0.01) when compared to vehicle-injected controls. Furthermore, the time course of behavioral suppression was similar in oPRL-treated animals to that observed in pars distalis-grafted males, with both groups showing the onset of deficits in sexual activity within 8 to 9 days from the induction of the hyperprolactinemic state. The similarity in pattern and time to onset of behavioral suppression in pituitary-grafted and oPRL-treated animals suggests that behavioral deficits observed in animals with pituitary grafts result from chronic elevation of serum PRL levels.     

Cultured cells from renal cortex of hibernators and nonhibernators. Regulation of cell K+ at low temperature

Cells were grown as primary monolayer cultures from kidney cortex of guinea pigs (nonhibernators), hamsters and ground squirrels (both hibernating species). When plates of cells were placed at 5 °C, cells of guinea pigs lost 37% of their K⁺ in 2 h and those of the hibernator lost about 10%.Uptake of ⁴²K into the cells exhibited a simple, single exponential time course at both temperatures. Unidirectional efflux of K⁺ was equal to K⁺ influx in all cultures at 37 °C and, within limits of error, in hibernator cells at 5 °C. Efflux was 3- to 5-fold greater than influx in guinea pig cells at 5 °C.After 2 h in the cold the ouabain-sensitive K⁺ influx remaining (7–15% of that at 37 °C) was about the same in the cells of the 3 species. Cells from active hamsters and from hibernating ground squirrels, however, exhibited significantly greater pump activity after 45 min in the cold (19 and 14%, respectively). The stimulation of K⁺ influx by increasing [K⁺]_o did not show an increase in K_m⁺ at 5 °C in cells of guinea pigs and ground squirrels. Lowering [K⁺]_c and/or raising [Na⁺]_c by treatment in low- and high-K⁺ media caused only slight stimulation of K⁺ influx, except in cells of ground squirrels at 5 °C in which the stimulation was at least 11-times greater than at 37 °C or in cells of guinea pigs at either temperature.This altered kinetic response of K⁺ transport to cytoplasmic ion stimulation with cooling accounted for about one-third of the improved regulation of K⁺ at 5 °C in ground squirrel cells; the other two-thirds was attributable to a greater decrease in K⁺ leak with cooling. The inhibition of active transport by cold in all 3 species was much less severe than that previously seen in any (Na⁺ + K⁺)-ATPase of mammalian cells.     

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient.

The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.     

Differentiation of chloroplast lamellae: Light harvesting efficiency and grana development

Incomplete development of chloroplast lamellae occurred when etiolated pea plants were greened under cycles of 2 min light, 118 min dark. Although the plastids had full photochemical activities, they were nearly agranal. They were also characterized by a high quantum requirement for whole chain electron transport in low light; this is thought to be the result of unequal light absorption by incompletely developed light-harvesting assemblies of photosystem I and II and a lack of regulation of excitation energy distribution between the two photosystems. Continuous illumination induced the final stages of membrane differentiation. These stages were primarily characterized by the appearance of grana stacking and an increase in photosynthetic unit size. A biphasic decrease in quantum requirement for whole chain electron transport correlated directly with the appearance of grana during the final steps of membrane assembly. Structural organization of the membrane may be related to the light-harvesting efficiency of the membrane.     

Selective phosphorylation of myosin light chain in intact skeletal muscle

The phosphorylation of myosin light chain was quantitated in fast and slow chicken skeletal muscles and in frog sartorius and semitendinosus muscles. The phosphate content of light chain was determined either as moles [³²P]phosphate per mole of light chain in ³²P-labeled muscles or as percentage phosphorylated light chain of the total P-light chain, measured by densitometry after separating the phospho and dephospho forms of P-light chain with two-dimensional gel electrophoresis. Both methods revealed that the percentage of total P-light chain which was phosphorylated did not exceed 50% either in maximally tetanized or caffeine-contracted skeletal muscle. This suggests that one of the two P-light chains is selectively phosphorylated in skeletal muscle.     

Identification of a rabbit B cell alloantigen with an antiserum produced by alloimmunization with thymus cells.

Alloimmunization with rabbit thymus cells resulted in an antiserum (anti-Rly) which was shown to react with rabbit lymphocytes by an indirect rosette technique. The titration curve obtained with dilutions of anti-Rly antiserum on lymph node cells revealed two plateaus indicating that the antiserum was multispecific; at low dilutions of antiserum, within the first plateau, both B and T cells were rosetted whereas at high dilutions, within the second plateau, only B cells were rosetted. The antigen detected at high dilution was designated LB-1 (lymphocyte B cell alloantigen 1). The evidence that the cells identified within the second plateau are B cells is as follows: 1) simultaneous enumeration of LB-1⁺ and Ig⁺ (B) cells by use of distinguishable erythrocytes (sheep and human) as indicator cells revealed that of the 53% rosettes observed, essentially all (51%) were mixed rosettes containing both erythrocytes whereas simultaneous enumeration of LB-1⁺ and T⁺ cells (identified by anti-T cell antiserum) showed essentially no mixed rosettes (less than 2%); 2) approximately 80% of purified Ig⁺ (B) cells were identified as LB-1⁺ cells whereas essentially no (< 1%) purified T cells could be detected as LB-1⁺; 3) the percentages of LB-1⁺ cells and Ig⁺ cells were both reciprocal to the precentages of T⁺ cells identified in various lymphoid organs except for bone marrow; 4) the removal of LB-1⁺ cells from spleen cells of rabbits immunized with sheep red blood cells resulted in a depletion (42–71%) of direct plaque forming cells (PFC). Since the percentages of bone marrow cells rosetted using anti-LB-1 antiserum (approximately 70%) was much greater than the percentage rosetted using anti-Ig (approximately 10%), it appears that the anti-LB-1 antiserum is not directed against an Ig allotype. The titration curves of the anti-Rly antiserum on peripheral blood lymphocytes of a large rabbit family suggested that the LB-1 antigen on B cells is an alloantigen probably inherited in simple Mendelian fashion. Adsorption studies indicated that the LB-1 antigen on B cells is not detectable on brain, liver, kidney or erythrocytes.     

Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: a model for immunodeficiency disease.

The inherited absence of either adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with severe immunological impairment. We have developed a cell culture model using a mouse T cell lymphoma to simulate ADA deficiency and to study the relationship between purine salvage enzymes and immune function. 2′-deoxyadenosine triphosphate (deoxyATP) levels have been shown to be greatly elevated in erythrocytes of immunodeficient, ADA-deficient patients, suggesting that deoxyadenosine is the potentially toxic substrate in ADA deficiency. Using a potent ADA inhibitor, we have demonstrated that deoxyadenosine is growth-inhibitory and cytotoxic to S49 cells, and that deoxyATP accumulates in these cells. Cell variants, unable to transport or phosphorylate deoxyadenosine, are much less sensitive to deoxyadenosine, indicating that intracellular phosphorylation of deoxyadenosine is required for the lethal effects.We have partially reversed the cytotoxic effects of deoxyadenosine with deoxycytidine in wild-type cells, but we cannot show any reversal in cell lines lacking deoxycytidine kinase. Adenosine (ado) kinase-deficient cells are extremely resistant to deoxyadenosine in the presence of deoxycytidine. This deoxycytidine reversal of deoxyadenosine toxicity is consistent with an inhibition of ribonucleotide reductase by deoxyATP, and we have shown that incubation of S49 cells with deoxyadenosine markedly reduces intracellular levels of deoxyCTP, deoxyGTP and TTP.Kinetics data in wild-type cells and in cell variants are consistent with the presence of two deoxyadenosine-phosphorylating activities — one associated with ado kinase and another associated with deoxycytidine kinase.The S49 cells appear to be a valid model for the simulation of ADA deficiency in cell culture, and from our results, we can suggest administration of deoxycytidine as a pharmacological regimen to circumvent the clinicopathologic symptoms in ADA deficiency.     

Sound localization by the Hawaiian squirrelfishes, Myripristis berndti and M. argyromus

Sound localization was investigated in a large pond open to a bay and similar to the normal environment of the animals. Observations were made of fish movements towards one of two underwater loud-speakers emitting squirrelfish alarm calls normally produced in response to predators. When the sound source was within 2·0 m of the test cage housing the fish, the subjects faced and moved toward the speaker. The animals responded some of the time when the source was within 3·0 m but generally did not orient to the sound source when the speaker was beyond 3·0 m. Response loss was correlated with the fish being in the acoustic far-field. Possible cues which release and direct localization remain unknown, but include particle velocity information alone, or some change in particle velocity: pressure relationships.     

Retention of K at low temperature in mouse L cells before and after cold adaptation

When exposed to 5 °C for periods of 3–4 weeks, mouse L cells, grown as monolayer cultures, lose K more slowly than do diploid mammalian cells. Subcultures of mouse L cells previously exposed to low temperature for intermittent periods lose K more slowly than subcultures not previously cold exposed. The superior retention of K in the cold may account in part for the better survival in the cold of mouse L cells than of diploid cells, and of cold-conditioned L cells than of unconditioned L cells.     

A quantitative study of phagocytosis by hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria

Fresh hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria were exposed to known concentrations of Bacillus megaterium, Escherichia coli, and Staphylococcus aureus in vitro and it was ascertained that all four types of cells of C. virginica and all three types of M. mercenaria became associated with the bacteria. Association is defined as either the first, i.e., contact and adherence, or second, i.e., engulfment, phase of phagocytosis. However, when the surfaces of each type of cell, as well as the percentages of each type in whole hemolymph, from both species of molluscs are taken into consideration, it is concluded that the granulocytes are the most important from the standpoint of phagocytosis.When hemocytes of M. mercenaria were exposed to Bacillus megaterium at 4°, 22°, and 37°C, it was found that the association indices were higher at the latter two temperatures. It is postulated, because of the results of Feng and Feng (1974), that nonself materials adhere with less frequency at 4°C and hence are not phagocytosed at this lower temperature.     

Characterization of [3H] corticosterone binding activity in adrenal incubation media

Glucocorticoid-binding activity in adrenal incubation media was investigated with regard to characterization of a protein-like ligand. Scatchard analysis of corticosterone binding activity indicated the presence of a single non-interacting protein with a dissociation constant (Kd) of 8.81 × 10?¹⁰ M (0°C), a value which is different from that of plasma and cytoplasmic glucocorticoid binding proteins. In addition, an observed lack of affinity of the protein for dexamethasone distinguishes the protein from Type II cytoplasmic receptor proteins. Thus our data suggest a glucocorticoid-binding protein which is distinct from the two known groups of glucocorticoid-binding proteins, corticosteroid-binding globulin (CBG) and cytoplasmic receptors.     

Alterations in uterine and placental prostaglandin F and E with gestational age in the rat

Experiments were designed to determine the chronological alterations in placental and uterine prostaglandin F and E (PGF and PGE) during pregnancy in the rat. Pregnant rats (sperm in the vagina = day 0) were sacrified at days 15, 18,19, 20, 21 and delivery (day 21 ) and placental and uterine tissues assayed (RIA) for PGF and PGE immediately (“”) or after 1 hour incubation (“”). Uterine content of PGF and PGE (ng PG/mg DNA) was increased significantly by day 19 and further increases were seen through delivery. Incubation of uterine tissue resulted in enhanced net production of PGF and PGE (p <.05) per mg DNA (as judged by tissue content and release into the incubation medium) by day 18 of pregnancy vs. day 15. Net production peaked around the time of delivery thus paralleling the alterations in tissue content .By contrast, no differences with gestational age were found in placental content of PGF and PGE , the concentrations throughout late gestation remaining in the range of uterine PGs at day 15. However, production of PGs per mg placental DNA increased markedly during incubation with significant enhancement detected by day 19 vs. 15, achieving levels even greater than the uterus .The and findings for the uterus are consistent with the hypothesis that increases in uterine PGs levels at the end of pregnancy may play an important role in parturition. The experiences with placental tissue suggest that the potential for PG production per placental cell may also increase in late gestation and thereby contribute to the augmented intrauterine availability of PGs at that time.     

Uptake of the yolk protein, lipovitellin, by developing crustacean oocytes

A variety of cytochemical techniques were used to demonstrate how crustacean lipovitellin accumulates within the egg. It was found that a protein serologically identical to the lipovitellin of yolk spheres was present in the hemolymph of vitellogenic crustaceans, but was absent from the hemolymph of males and immature females.In the three crustacean species studied (Uca pugilator, Cambarus clarkii, and Libinia emarginata), pinocytosis of fluorescein-conjugated lipovitellin and trypan blue occurred only during those periods when oocytes were accumulating yolk.It may be concluded from the present studies that yolk spheres develop in crustacean eggs primarily through micropinocytotic uptake of lipovitellin from the hemolymph, although other oocyte proteins appear to be made in the oocyte.     

Regulation of lipogenesis in adipose tissue: the significance of the activation of pyruvate dehydrogenase by insulin

Simultaneous measurements were made of lipogenesis and pyruvate dehydrogenase activity in segments of rat epididymal adipose tissue incubated with saturating amounts of [U-¹⁴C]glucose and insulin. Glucose was converted to fatty acids at a rate only 64–79% of that permitted by the tissue's content of the active form of pyruvate dehydrogenase (PDH_a). Addition of either of the electron acceptors, phenazine methosulfate (10 μm) or N,N,N′,N′-tetramethyl-p-phenylenediamine (50 μm), increased lipogenesis until it equalled the PDH_a activity of the tissue. Pyruvate release was increased 2-fold or more by the electron acceptors, suggesting that the increase in lipogenesis might have resulted from an increase in the intracellular pyruvate levels such that PDH_a became saturated with substrate. Higher levels of the electron acceptors decreased PDH_a activity, and reduced lipogenesis correspondingly. The data suggest that the maximal rate of lipogenesis in the presence of glucose and insulin is limited by the inability of the tissue to elevate pyruvate levels sufficiently to saturate PDH_a. Although glycerol release was increased by either electron acceptor and insulin partially overcame this effect, the effects of the electron acceptors on PDH_a activity could not be attributed to an increase in lipolysis.     

Occurrence of reiterated sequences in an untranslated region of Simian virus 40 DNA determined by nucleotide sequence analysis.

An earlier report (Subramanian, Dhar, and Weissman, 1977c) presented the nucleotide sequence of Eco RII-G fragment of SV40 DNA, which contains the origin of DNA replication. The nucleotide sequence of Eco RII-N fragment located next to Eco RII-G on the physical map of SV40 DNA is presented in this report. Eco RII-N is found to be a tandem duplication of the last 55 nucleotides of Eco RII-G. This tandem repeat is immediately preceded by two other reiterated sequences occurring within Eco RII-G, one of them being a tandem repeat of 21 nucleotides and the other a nontandem repeat of 10 nucleotides. These repetitive sequences occur in close proximity to the origin of DNA replication which is known to contain other specialized sequences such as a few palindromes (one of which is 27 long and possesses a perfect 2-fold axis of symmetry), one "true" palindrome, and a long A/T-rich cluster. The repeats (and the replication origin) occur within an untranslated region of SV40 DNA flanked by (the few) structural genes coding for the "late" proteins on the one side and that (those) coding for the "early" protein(s) on the other side. The reiterated sequences are comparable in some respects to repetitive sequences occurring in eucaryotic DNAs. Possible biological functions of the repeats are discussed.     

Steady state kinetic mechanism of the Escherichia coli coenzyme A transferase.

Developing chloroplasts isolated from greening cotyledons and isolated etioplasts were capable of synthesizing and accumulating Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins when incubated in the dark in the presence of protoporphyrin and cofactors. These results constituted the first unambiguous demonstration of the insertion of magnesium into exogenous protoporphyrin in a cell-free system from higher plants. The metalloporphyrin synthetic activity did not occur in the absence of the plastids or when the plastids were heated in a 100 °C water bath for 2 min. It is thus suggested that, in higher plants, the in vitro insertion of magnesium into protoporphyrin is an enzymatic reaction.     

Ornithine decarboxylase and polyamines in the resumption of cycling by diluted and reoxygenated mammalian cells

Plateau-phase V79 Chinese hamster cells induced to reenter the proliferative cell cycle from G1/G0 by dilution in fresh medium showed an early increase in ornithine decarboxylase (ODC) followed by increases in some polyamines and in DNA synthesis. In contrast, cells accumulated in G1/G0 by growth in extreme hypoxia for 60 h and induced to recycle by reoxygenation did not respond with early increases in either ODC or polyamines.     

Biosynthesis of adenosyl-d-methionine and adenosyl-2-methylmethionine by Candida utilis

Supplementation of the culture medium of Candida utilis with d-methionine or 2-methyl-dl-methionine leads to intracellular synthesis of S-adenosyl-d-methionine and S-adenosyl-2-methylmethionine. The identity of the sulfonium compounds was established by tracer technique, chromatography, acid hydrolysis, and examination of the released methionine and 2-methylmethionine. In addition to the expected sulfur amino acid component, both adenosine sulfonium fractions contained S-adenosyl-l-methionine. This is explained by transmethylation of S-adenosyl-d-methionine and of S-adenosyl-2-methyl-methionine with endogenous l-homocysteine; the resulting l-methionine reacts with ATP to form S-adenosyl-l-methionine. Experiments with purified cell-free preparations of S-adenosylmethionine synthetase (EC 2.5.1.6) from C. utilis confirmed the reaction of ATP with d-methionine or 2-methyl-dl-methionine.