SPERMATOGONIAL STEM CELL RENEWAL IN THE MOUSE: II AFTER CELL LOSS

The repair of the mouse seminiferous epithelium after cell loss has been studied in seminiferous tubules mounted in toto . Cell loss was inflicted by injection of Myleran in a dose of 10 mg/kg body weight. In stages 7–8, in which we mainly counted, the numbers of A_isolated (A_is), A_paired (A_pr), A_aligned (A_al) and A₁ spermatogonia and resting primary spermatocytes decreased after injection. After about 24 days normal numbers of A₁ spermatogonia were found again. Thereafter a substantial overshoot in the number of A₁ spermatogonia was found.
While normally most of the A_pr and A_al cells differentiate into A₁ spermatogonia in stages 3 and 4 and do not divide until stage 9, during repair they pass through one more division during stages 6 and 7. Normally, during these stages divisions of these spermatogonia are rare. Owing to this extra division the transformation of A_pr and A_al into A₁ spermatogonia is delayed from stage 3 or 4 to stage 8, i.e. still before stage 9, in which A₁ spermatogonia divide. From 16 days after the injection onwards the extra division takes place less generally and more and more cells transform into A₁ spermatogonia at the normal time.     

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.     

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.     

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.     

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.     

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.     

Free and conjugated gibberellins in dormancy and germination of apple seeds

Halińska, A. and Lewak, St. 1987. Free and conjugated gibberellins in dormancy and germination of apple seeds.
The presence of gibberellin A₄ (GA₄) was confirmed in partly stratified seeds of apple ( Malus domestica Borb., cv. Antonówka) by mass spectrometry of the methyl ester. Levels of free and conjugated gibberellins A₄₊₇ and A₉ changed during drying of mature seeds, during cold and warm stratification, as well as during germination of dormant and non-dormant embryos. The temporary rise in GA₄₊₇ during cold stratification and during the culture of dormant embryos as well as the lack of it under conditions of warm stratification, allowed us to postulate a role for GA₄₊₇ in the removal of dormancy. In addition, GA₉ was absent in dormant embryos and increased during cold stratification and during the culture of non-dormant embryos. This suggests the involvement of GA₉, in induction of normal development of the seedling. The equivalence between changes in free and conjugated GAs suggests that formation and hydrolysis of conjugates are involved in the control of the physiologically active levels of free GA₄₊₇ and GA₉.     

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.     

Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle

Abstract. Multivariate analysis of the expression of cyclin proteins and DNA content has opened new possibilities for the study of the cell cycle. By virtue of their cell cycle phase specificity, the expression of cyclins may serve, in addition to DNA content, as another marker of a cell's position in the cycle, and provide information about the proliferative potential of cell populations. Several applications of the methodology based on bivariate analysis of DNA content v . expression of B, E and D type cyclins are reviewed: 1 expression of cyclins by individual cells during their progression through the cycle can be studied, using exponentially growing cells without the necessity of cell synchronization or other perturbations of the cycle; 2 cells having the same DNA content but residing in different phases of the cycle (e.g. G₂ diploid v. G₁ tetraploid) can be distinguished; 3 cell transition from G₀ to G₁ and progression through G₁ (e.g. mitogen stimulated lymphocytes) can be assayed; 4 the population of proliferating cells can be distinguished from noncycling cells based on dual cell labelling with a G₁ and G₂ cyclin antibody; 5 cyclin restriction points can serve as additional cell cycle landmarks to map the point of action of antitumour drugs; 6 unscheduled expression of cyclins (e.g. the presence of cyclin B1 during G₁ and S) can be detected in several tumour transformed cell lines, possibly indicating disregulation of the machmery of cell cycle progression. The last finding 6 is of special importance, because such disregulation may be of prognostic consequence in human tumours.     

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 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.     

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.     

Chromatography of acid phytohormones on columns of Sephadex LH-20 and insoluble poly-N-vinylpyrrolidone, and application to the analysis of conifer extracts

The chromatographic behaviour of abscisic acid (ABA), indole-3-acetic acid (IAA), phenylacetic acid (PAA), and gibberellins A₁, A₄, A₈, A₉, A₁₃ and A₂₀ on columns of Sephadex LH-20 and insoluble poly- N -vinylpyrrolidone (PVP) eluted with buffers of different pH values is described. PVP shows considerable batch differences that must be carefully checked. Chromatography of acidic ethyl acetate-soluble fractions of Scots pine ( Pinus sylvestris L.) extracts at pH 4.5 resulted in great losses of phytohormones, due to poor solubility of the extracts. If the extracts were applied to the column dissolved in buffer of pH 7.5, subsequent elution at pH 4.5 was possible with only small losses. The performance of the chromatographic column was strongly affected by the application volume. A combined PVP/Sephadex LH-20 column eluted at pH 4.5 allows remarkable purification of pine and spruce ( Picea abies (L.) Karst.) extracts, collection of IAA in a fraction that can be directly analyzed by e.g. the indolo-α-pyrone method, and collection of another fraction containing ABA, PAA and probably most of the known C₁₉-gibberellins; whereas the C₂₀-gibberellin A₁₃ is eluted later (with IAA).     

Adenosine A2b Receptors Mediate an Increase in Interleukin (IL)-6 mRNA and IL-6 Protein Synthesis in Human Astroglioma Cells

Abstract: The cytokine interleukin (IL)-6 has recently been demonstrated to play a role in the pathology of Alzheimer's disease (AD). The mechanisms leading to increased IL-6 levels in brains of AD patients are still unknown. Because in experimental animals ischemia increases both the level of cytokines and the extracellular concentrations of adenosine in the brain, we hypothesized that these two phenomena may be functionally connected and that adenosine might increase IL-6 gene expression in the brain. Here we show that the mixed A₁ and A₂ agonist 5'-( N -ethylcarboxamido)adenosine (NECA) induces an increase in IL-6 mRNA levels and protein synthesis in the human astrocytoma cell line U373 MG. The A₁-specific agonists R -phenylisopropyladenosine and cyclopentyladenosine are much less potent, and the A_2a-specific agonist CGS-21680 shows only marginal effects. Increased levels of mRNA are already found within 30 min after NECA treatment. The A_2a-selective antagonists 8-(3-chlorostyryl)caffeine and KF17837 [( E )-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine], which have also some antagonistic properties at A_2b receptors, and the nonspecific adenosine antagonist 8-phenyltheophylline were equipotent at inhibiting the NECA-induced increase in IL-6 protein synthesis, whereas the specific A₁ antagonist 8-cyclopentyl-1,3-dipropylxanthine is much less potent. The results indicate that adenosine A_2b receptors participate in the regulation of the IL-6 gene in astrocytoma cells.     

Impaired spermatogenesis in the Japanese eel, Anguilla japonica: Possibility of the existence of factors that regulate entry of germ cells into meiosis

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations revealed that HCG-injected eels could be classified into three types based on their testicular conditions. Type 1 eels had a well-developed testis and the milt could be acquired by hand-stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand-stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11–ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced by HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence of 2³ to 2⁶ late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into meiosis.     

The testicular cycle of Gambusia affinis holbrooki (Teleostei: Poeciliidae)

Fifteen male mosquito fish ( Gambusia affinis holbrooki ) were collected in 1989 on the 15th of each month to perform a quantitative histologic study of the annual testicular cycle including a calculation of the gonadosomatic index, testicular volume, and the total volume per testis occupied by each germ cell type. The cycle comprises two periods: spermatogenesis and quiescence. The spermatogenic period begins in April with the development of primary spermatogonia into secondary spermatogonia, spermatocytes and round spermatids. In May, the first spermatogenic wave is completed and the testicular volume begins to increase up to June when the maximum testicular volume and gonadosomatic index are reached. Germ cell proliferation with successive spermatogenetic waves continues until August. In September germ cell proliferation ceases and neither secondary spermatogonia nor spermatocytes are observed. However, spermiogenesis continues until October. In November, spermiogenesis has stopped and the testis enters the quiescent period up to April. During this period only primary spermatogonia and spermatozoa are present in the testis. In addition, a few spermatids whose spermiogenesis was arrested in November are observed. Testicular release of spermatozoa is continuous during the entire spermatogenesis period. The spermatozoa formed at the end of this period (September-October) remain in the testis during the quiescent period and are released at the beginning of the next spermatogenesis period in April. Developed Leydig cells appear all year long in the testicular interstitium, mainly around both efferent ducts and the testicular tubule sections showing S₄ spermatids.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

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.