CELL CYCLE PROPERTIES OF DIFFERENTIATING SPERMATOGONIA IN ADULT SPRAGUE-DAWLEY RATS

The duration of the mitotic cycle and of its components was analysed for each of the six successive generations of differentiating spermatogonia (A₁, A₂, A₃, A4, intermediate and B), using radioautographed whole mounts of seminiferous tubules from testes of adult Sprague-Dawley rats. Cell cycles were determined from two successive waves of per cent labeled metaphases obtained during the period of 81 hr after a single dose of ³H-thymidine. Except for the A₁ spermatogonia, all spermatogonial types (A₂ to B) had similar cell cycle durations of 41-42.5 hr and comparable pre-DNA synthesis phases (G₁) of 11-13 hr. Although the combined duration of DNA synthesis (S) and the post-synthesis phase (G₂) remained identical for all the cell types including A₁, there was a progressive lengthening of the S period at the expense of G₂ during the process of spermatogonial maturation. This change was most marked during the transition from A₁ to A₃ spermatogonia when the S period increased from 14 hr to 21 hr, and the G₂ phase shortened from 13 hr to 7.5 hr. This feature seems to be unique to germ cells and may be associated with an increasing amount of heterochromatin in the nucleus. Excluding the development of type A₁ cells, the entire process of spermatogonial maturation lasted for 208 hr. Combined data on cell cycle times indicated that every 313 hr or 13 days, a new sequence of spermatogonial differentiation was initiated by the A₁ cells. This was equivalent to the duration of one 'cycle' of the seminiferous epithelium as measured by other techniques.     

A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse

In whole mounts of seminiferous tubules of C3H/101 F₁ hybrid mice, spermatogonia were counted in various stages of the epithelial cycle. Furthermore, the total number of Sertoli cells per testis was estimated using the disector method. Subsequently, estimates were made of the total numbers of the different spermatogonial cell populations per testis.

The results of the cell counts indicate that the undifferentiated spermatogonia are actively proliferating from stage XI until stage IV. Three divisions of the undifferentiated spermatogonia are needed to obtain the number of A₁ plus undifferentiated spermatogonia produced each epithelial cycle. Around stage VIII almost two-thirds of the A_pr and all of the A_al spermatogonia differentiate into A₁ spermatogonia. It was estimated that there are 2.5 × 10⁶ differentiating spermatogonia and 3.3 × 10⁵ undifferentiated spermatogonia per testis. There are about 35,000 stem cells per testis, constituting about 0.03% of all germ cells in the testis. It is concluded that the undifferentiated spermatogonia, including the stem cells, actively proliferate during about 50% of the epithelial cycle.     

Gibberellins in relation to flowering in Polianthes tuberosa

Endogenous gibberellins (GAs) in corms of Polianthes tuberosa L. (cv. Double) were isolated and identified by high performance liquid chromatography, bioassay and combined capillary gas chromatography-mass spectrometry (GC-MS). Gibberellins A₁, A₁₉, A₂₀ and A₅₃ were quantified at the vegetative, early floral initiation and flower development stages. The identification of 13-hydroxylated GAs indicates the presence of the early 13-hydroxylation pathway in P. tuberosa corms. An increase in GA₁ and GA₂₀, and a decrease in GA₁₉ levels, coincided with the transition from the vegetative phase to the stages of early floral initiation and flower development. GA₅₃ stayed at constant levels at the 3 different growth stages. The absence of GA₁ in vegetative corms and its presence in corms at early floral initiation and flower development stages suggest that GA₁ is a causal factor in inducing floral initiation in P. tuberosa . When GA₁, GA₃, GA₄, GA₂₀ and GA₃₂ were applied to corms at the vegetative stage (plants about 5 cm in height), floral initiation was promoted by all of the GAs used, GA₃₂ being the most active. In contrast with the other GAs, GA₃₂ had no effect on stem elongation. Therefore, it is suggested that hydroxylated C-19 GAs play an important role in flower induction in P. tuberosa .     

Rat-1 Fibroblasts Engineered with GAD65 and GAD67 cDNAs in Retroviral Vectors Produce and Release GABA

Abstract: The effects of the adenosine A₁ agonist N ₆-cyclohexyladenosine (CHA) on MPTP-induced dopamine (DA) depletion in the striatum of C57BL/6 mice were studied. Twenty hours after a single injection of MPTP (30 mg/kg, s.c.), the toxin caused 62% depletion of striatal DA. CHA (0.2–3 mg/kg, s.c.), when given together with MPTP, prevented the toxin-induced DA depletion in a dose-dependent manner. This protective action was apparently mediated by the A¹ receptors, because this effect was selectively antagonized by pretreating the animals with the A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg, i.p.) but not with the A₂ antagonist 1,3-dipropyl-7-methylxanthine (25 mg/kg, i.p.). When CHA (3 mg/kg) was injected 5 h after MPTP administration, at which point striatal DA levels were already reduced significantly, a rapid and complete recovery of the striatal DA levels occurred. These neurochemical data suggest that the A₁ agonist CHA is potentially useful as a neuroprotective agent against MPTP-induced toxicity.     

Effect of glutamate intake during gestation on adenosine A(1) receptor/adenylyl cyclase pathway in both maternal and fetal rat brain

Pregnant Wistar rats were orally treated with 1 g/L l -glutamate during the entire gestational period and the status of adenosine A₁ receptor (A₁R)/adenylyl cyclase transduction pathway from maternal and fetal brain was analyzed. Glutamate consumption, estimated from the loss of water from the drinking bottles, was 110 ± 4.6 mg/kg/day. In mother brains glutamate intake did not significantly alter the B _max value, although the K _d value was significantly decreased. However in fetus brain, a significant decrease in B _max was observed, without an alteration of K _d value. Similar results were observed by western blot assays using specific A₁R antibody, suggesting a down-regulation of A₁R in fetal brain. Concerning α subunits of inhibitory G proteins (Gi), αGi₃ protein was slightly but significantly decreased in maternal brain without alterations of either Gi₁ or Gi₂. In contrast, αGi₁ and αGi₂ isoforms were increased in fetal brain. On the other hand, basal, forskolin, and forskolin plus GTPγS-stimulated adenylyl cyclase activity was significantly decreased in both maternal and fetal brain, and this was more prominent in fetal than in maternal brain. Finally, A₁R functionality was significantly decreased in mother brain whereas no significant differences were detected in fetus brain. These results suggest that glutamate administered to pregnant rats modulates A₁R signaling pathways in both tissues, showing an A₁R down-regulation in fetal brain, and desensitization in maternal brain.     

THE SPERMATOGONIAL STEM CELL POPULATION IN ADULT RATS

Radioautographed whole mounted seminiferous tubules from adult rat testes were used to analyse undifferentiated type A spermatogonia at various intervals up to 81 hr following a single injection of ³H-TdR. the data obtained led to the identification of the spermatogonial stem cell and to the formulation of a new model for spermatogonial renewal and differentiation. Undifferentiated type A cells were morphologically alike, but were topographically classified as (1) isolated or (2) paired and aligned. Although labeled isolated A cells were scattered over most stages of the seminiferous epithelium, their proliferative activity varied with the stage; their labeling index was 20-30% in stages I and II, but less than 1% in stages VII and VIII. By tracing the labeled divisions of isolated A spermatogonia in time, it was seen that some daughter cells became separated from one another to form two new isolated cells, while others remained together as paired A spermatogonia. Analysis of two successive waves of labeled mitoses revealed that most paired A spermatogonia continued to proliferate forming four aligned A cells, many of which divided again to produce a chain of eight and so on. the greatest incidence of labeling among paired and aligned A spermatogonia occurred in stages XIII-III. In stage I, where the labeling index was 50%, the calculated proliferative fraction was 1 for these spermatogonia. Between stages II and V, they began to leave mitotic cycle, and during stage V this entire cohort morphologically transformed into A₁ spermatogonia. Labeled metaphase curves for undifferentiated A spermatogonia were distinct from any of the curves previously constructed for the six classes of differentiating spermatogonia, especially because of particularly long S and G₂ phases in the former. the cell cycle time of paired and aligned A cells was 55 hr, compared to an average of 42 hr for differentiating types A₂ to B.     

Modulation of adenosine A1 and A2A receptors in C6 glioma cells during hypoxia: involvement of endogenous adenosine

During hypoxia, extracellular adenosine levels are increased to prevent cell damage, playing a neuroprotective role mainly through adenosine A₁ receptors. The aim of the present study was to analyze the effect of hypoxia in both adenosine A₁ and A_2A receptors endogenously expressed in C6 glioma cells. Two hours of hypoxia (5% O₂) caused a significant decrease in adenosine A₁ receptors. The same effect was observed at 6 h and 24 h of hypoxia. However, adenosine A_2A receptors were significantly increased at the same times. These effects were not due to hypoxia-induced alterations in cells number or viability. Changes in receptor density were not associated with variations in the rate of gene expression. Furthermore, hypoxia did not alter HIF-1α expression in C6 cells. However, HIF-3α, CREB and CREM were decreased. Adenosine A₁ and A_2A receptor density in normoxic C6 cells treated with adenosine for 2, 6 and 24 h was similar to that observed in cells after oxygen deprivation. When C6 cells were subjected to hypoxia in the presence of adenosine deaminase, the density of receptors was not significantly modulated. Moreover, DPCPX, an A₁ receptor antagonist, blocked the effects of hypoxia on these receptors, while ZM241385, an A_2A receptor antagonist, was unable to prevent these changes. These results suggest that moderate hypoxia modulates adenosine receptors and cAMP response elements in glial cells, through a mechanism in which endogenous adenosine and tonic A₁ receptor activation is involved.     

Adenosine A1 Receptors in Cultured Cerebellar Granule Cells: Role of Endogenous Adenosine

Abstract: Adenosine A₁ receptors as well as other components of the adenylate cyclase system have been studied in cultured cerebellar granule cells. No significant changes in adenosine A₁ receptor number, assayed by radioligand binding in intact cells, were detected from 2 days in vitro (DIV) until 7 DIV. Nevertheless, a decline in this parameter was detected at 9 DIV. The steady-state levels of α-G_s and α-G_i, detected by immunoblotting, showed similar profiles, increasing from 2 to 5 DIV and decreasing afterward. Forskolin-stimulated adenylate cyclase levels also showed an increase until 5 DIV, decreasing at 7 and 9 DIV. The adenosine A₁ receptor analogue cyclopentyladenosine (CPA) was able to inhibit cyclic AMP accumulation at 2, 5, and 7 DIV but failed to do so at 9 DIV. This inhibition was prevented by the specific adenosine A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The presence of adenosine deaminase in the culture increased adenosine A₁ receptor number during the period studied and induced recovery of the inhibitory effect of CPA, lost after 7 DIV. These data suggest that functional expression of adenosine A₁ receptors and the other components of the adenylate cyclase system is subjected to regulation during the maturation of cultured cerebellar granule cells and demonstrates a key role for endogenous adenosine in the process.     

Effects of prohexadione (BX-112) and gibberellins on shoot growth in seedlings of Salix pentandra

Effects of gibberellins A₁, A_4/7, A₉, A₁₉ and A₂₀ and growth retardants were studied on shoot elongation in seedlings of Salix pentandra L. The growth-retarding effects of CCC and ancymidol were antagonized by all the gibberellins tested. The novel plant growth regulator prohexadione (free acid of BX-112), which is suggested to block 3β-hydroxylation of gibberellins, effectively prevented shoot elongation in seedlings grown under long photoperiod. Initiation of new leaves was only slightly reduced. GA₁, but not GA₁₉ and GA₂₀, was active in overcoming the inhibition of stem elongation of seedlings, treated with prohexadione, GA₁₉, GA₂₀ and GA₁ are native in S. pentandra , and the results are compatible with the hypothesis that GA₁ is active per se in shoot elongation, and that the effect of GA₁₉ and GA₂₀ is dependent on their conversion to GA₁.
A mixture of GA₄ and GA₇ was as active as GA₁ in promoting shoot elongation in seedlings treated with prohexadione, while GA₉ showed slight activity only when applied at high doses.     

Chronic Exposure to Adenosine Receptor Agonists and Antagonists Reciprocally Regulates the A1 Adenosine Receptor-Adenylyl Cyclase System in Cerebellar Granule Cells

Abstract: Chronic treatment with the adenosine receptor antagonist caffeine evokes an up-regulation of A₁ adenosine receptors and increased coupling of the receptor to G proteins in rat brain membranes. However, chronic agonist exposure has not been explored. Primary cultures of cerebellar granule cells were exposed chronically to A₁ adenosine receptor agonists and antagonists. Exposure to the A₁ adenosine receptor agonist N ⁶-cyclopentyladenosine resulted in (1) a time- and concentration-dependent reduction in the density of receptors labeled by 1,3-[³H]dipropyl-8-cyclopentylxanthine, (2) an enhanced ability of guanyl nucleotides to decrease the fraction of A₁ adenosine receptor sites displaying high affinity for 2-chloroadenosine, and (3) a functional uncoupling of receptors from adenylyl cyclase (EC 4.6.1.1). The adenosine antagonists caffeine and 8- p -sulfophenyltheophylline produced alterations in A₁ adenosine receptor homeostasis that were antipodal to those associated with agonist treatment. Antagonist exposure (1) increased the density of A₁ adenosine receptors in cerebellar granule cell membranes, (2) blunted the effect of guanyl nucleotides on receptor coupling to G proteins, and (3) increased the functional coupling of receptors to adenylyl cyclase inhibition. Forskolin treatment of cerebellar granule cells did not affect receptor density, suggesting that cyclic AMP is not involved in the regulation of A₁ adenosine receptor expression.     

Activation of Ethanolamine Phospholipase A2 in Brain During Ischemia

Abstract: Extracts of acetone-dried powders from ischemic gerbil brain were examined for phospholipase A₁ and A₂ activities with phosphatidylethanolamine at pH 7.2. Ischemia was induced by bilateral ligation, and the animals were killed by immersion into liquid nitrogen. Bilateral ligation with ketamine as general anesthetic resulted in a rapid, transient increase in phospholipase A₂ activity. The activity increased from 0.46 nmolihimg protein at 0 time to 0.82 nmol/h/mg protein at 1 min of ligation. Phospholipase A₁ activity also increased from 0.7 to 1.3 nmol/h/mg protein within the 1st min. When Nembutal was used as anesthetic, the phospholipase activation was earlier, within the first 30 s. Similar results were found for ischemia induced by decapitation of Wistar rats without anesthesia. Bilateral ligation of the carotid arteries of the gerbil is known to increase the concentration of free fatty acids, particularly arachidonate. This increase is, at least in part, due to phospholipase A activation. As ethanolamine phospholipase A₂ in brain does not require Ca²⁺ for activity, these results suggest that phospholipase A₂ activation in ischemic brain results from a covalent modification of the enzyme.     

Endogenous gibberellins in young seedlings of wheat (Triticum aestivum) cultivars

Gibberellins A₁, A₃, A₄ and A₇ were identified by combined gas chromatography mass spectrometry (GC-MS) in leaf and stem tissues of 17-day-old seedlings of wheat ( Triticum aestivum L. ), cvs Siete Cerros (semi-dwarf, Rht1) and Møystad (tall), of F₁, hybrids from the cross Møystad × Siete Cerros and of 2 selected lines from the cross Møystad x Sonora 64 (Rht1 and Rht2). GA, and GA, were identified by full scan mass spectra separately in all 5 extracts, GA₄ and GA₇, were identified by selected ion monitoring in a bulked fraction. About 90% of the biological activity (Tan-ginbozu dwarf rice bioassay) in all 5 extracts was due to the GA₁/GA₃-fraction.     

ON THE PHOSPHOLIPASE A2 ACTIVITY OF HUMAN CEREBRAL CORTEX

Abstract— Preparations of phospholipase Az have been obtained from human cerebral cortex. The enzyme was extracted from acetone-dried tissue and purified by heat-treatment and gel filtration on Sephadex.
Although heating at 65°C or 70°C destroys most of the phospholipase A₁ activity that is present in crude extracts, a small proportion remains associated with the A₂ activity during these procedures. The heat-treated extracts hydrolyse lecithin in preference to phosphatidyl-ethanolamine but have no action on lysolecithin or neutral lipids. The results suggest that A₂ activity and the heat-stable component of A₁ may both be due to a single phospholipase A that can hydrolyse diacylglycerophosphatides at either the 2-or the 1-position, to form a mixture of isomeric lysoderivatives.
A molecular weight of 55,000 was calculated for the enzyme.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE CELL PROGRESS FROM M TO G1 AND S STAGES IN CULTURED MOUSE LEUKEMIA L5178Y CELLS

Actinomycin D (0.5 μg/ml) did not prevent M stage cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The position of the block was approximately 1.4 hr before S stage or just after the beginning of G₁ stage. Actinomycin D in this concentration also significantly depressed uridine-³H uptake into G₁ stage cells, but did not suppress leucine-³H uptake by M and G₁ cells. This suggests that some proteins may be synthesized in M and G₁ stage cells by messenger RNA left over from the previous cell cycle. However, entry of G₁ cells into S stage would require synthesis of new messenger RNA near the beginning of G₁ stage. Puromycin (10 μg/ml) did not prevent M cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The site of blockage was about 0.7 hr before S stage or in the first two-third of G₁ stage. This might be the site where the cells synthesize new G₁ proteins necessary for entry to S stage.
Comparison of sensitivities of G₁ and G₂ stages to the two antibiotics reveals that the puromycin sensitivity of G₁ cells was similar to that of G₂ cells, but the actinomycin D sensitivity of G₁ was greater than that of G₂ cells.     

Desensitisation of the Adenosine A1 Receptor by the A2A Receptor in the Rat Striatum

Abstract: The influence of the adenosine A_2A receptor on the A₁ receptor was examined in rat striatal nerve terminals, a model for other cells in which these receptors are coexpressed. Incubation of striatal synaptosomes with the A_2A receptor agonist 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS 21680) caused the appearance of a low-affinity binding site for the A₁ receptor agonist 2-chloro- N ⁶-cyclopentyladenosine (CCPA). This effect was blocked by the A_2A receptor antagonist ZM241385 and by the protein kinase C inhibitor chelerythrine, but not by the protein kinase A inhibitor N -(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004). The effect was not seen with striatal membranes or with hypotonically lysed synaptosomes. These results demonstrate a protein kinase C-mediated heterologous desensitisation of the A₁ receptor by the A_2A receptor.     

Metabolism of tritiated gibberellin A1 in seedlings of Norway spruce, Picea abies

Five-week-old seedlings of Norway spruce, Picea abies (L.) Karst., metabolized 1,2-/³H/-gibberellin A₁ into a single major compound chromatographically similar to gibberellin A₈. The conversion rate exceeded 10% within the 24-h incubation period.     

Chronic Ethanol Reduces Immunologically Detectable Gqα/11α in NG108–15 Cells

Abstract: The amyloid protein (βA₄) is found in the CNS of patients with Alzheimer's disease; however, the pathogenic role of this protein is not known. In the present study, a peptide fragment of βA₄βA₄ 25–35; Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-NH₂), which contains the conserved C-terminal sequence of substance P (X-Gly-Leu-Met-NH₂), and the neuropeptide substance P (SP) were examined for their ability to modulate nicotine-evoked secretion from cultured bovine adrenal chromaffin cells. Secretion of the released endogenous catecholamines was monitored by electrochemical detection after separation by HPLC. Secretion induced by 10⁻⁵ M nicotine was inhibited by SP and βA₄ 25–35. The IC₅₀ of SP and βA₄ 25–35 was 3 × 10⁻⁶ and 3 × 10⁻⁵ M , respectively. SP and βA₄ 25–35 both protected against nicotinic receptor desensitization. However, βA₄ 25–35 was ∼ 10-fold less effective than SP in its protective effect. The present work shows that βA₄ 25–35 can mimic the modulatory actions of SP on the nicotinic response of cultured bovine chromaffin cells, i.e., inhibition of the nicotinic response and protection against nicotinic desensitization. These modulatory actions may be associated with changes in nicotinic receptor levels reported to occur in Alzheimer's disease.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.