Tripeptidil peptidasa 1 en pacientes con ceroidolipofuscinosis neuronal infantil tardía / Tripeptidyl peptidase 1 in patients with late infantile neuronal ceroid lipofuscinosis

Introducción: Las ceroidolipofuscinosis neuronales (CLN) representan un grupo de enfermedades lisosomales hereditarias de herencia autosómica recesiva, de presentación más frecuente durante la niñez, caracterizadas neuropatológicamente por acumulación de lipopigmentos autofluorescentes en los lisosomas de neuronas y otras células. Clínicamente se presentan con pérdida de las habilidades psicomotoras adquiridas, incoordinación motora, ataxia, pérdida de la visión, cambios de conducta, convulsiones de difícil tratamiento asociadas a mioclonías y una corta expectativa de vida. En la actualidad, se conocen 10 formas genéticamente distintas de esta enfermedad, entre ellas la forma infantil tardía donde las manifestaciones clínicas aparecen entre el segundo y cuarto año de vida. El gen responsable de la enfermedad es el TPP1 ubicado en 11p15 y codifica la enzima tripeptidil peptidasa 1. Pacientes y métodos: Se estandarizó la técnica para el diagnóstico enzimático de la ceroidolipofuscinosis neuronal infantil tardía a través de sangre seca en papel de filtro en 76 individuos sanos en edad preescolar y adulta de población venezolana. La actividad enzimática de la TPP1 fue determinada en 9 pacientes con diagnóstico clínico de ceroidolipofuscinosis infantil tardía 2 (CLN2). Resultados: Seis pacientes mostraron valores de actividad muy por debajo del rango establecido (0,11-0,45 nmol/mancha) para los controles sanos en edad preescolar, confirmando el diagnostico enzimático. Tres de los 14 padres estudiados presentaron valores en el rango de heterocigotos. Conclusiones: El diagnóstico enzimático de CLN2 a través de la determinación de la actividad enzimática de la enzima TPP1 mediante la técnica de sangre seca en papel de filtro permite un diagnóstico rápido, sencillo, económico y confiable(AU)

Introduction: Neuronal ceroid lipofuscinoses are a group of inherited autosomal recessive lysosomal diseases, most commonly found in infancy. These are neuropathologically characterised by accumulation of an autofluorescent lipopigment in neurons and other cells. This condition is clinically characterised by loss of motor and cognitive skills, lack of motor coordination, ataxia, progressive visual impairment, behavioural changes; seizures of difficult to manage seizures, particularly myoclonic, and premature death. Ten clinical forms have been described, one of which is late infantile where clinical signs begin between two and four years. The gene responsible for this disease is located at 11p15 locus, and the enzyme encoded by this gene is the tripeptidyl peptidase 1. Patients and methods: We standardised the technique for the enzymatic diagnosis of late infantile neuronal ceroid lipofuscinoses from dried blood on filter paper card in 76 healthy individuals adults and children in order to establish a normal range in the Venezuelan population. The tripeptidyl peptidase activity was also determined in 9 patients with a clinical diagnosis of late infantile neuronal ceroid lipofuscinoses. Results: Six of the samples showed activity lower than the lowest control value (0.11 to 0.45 nmol/spot) from healthy controls of infantile age, confirming the enzymatic diagnosis. Three of the 14 parent samples analysed showed values in the heterozygote ranges. Conclusions: The enzymatic diagnosis of late infantile neuronal ceroid lipofuscinoses from dried blood on filter paper card is a rapid, easier, less expensive and accurate molecular diagnosis tool(AU)

In vitro cytochemical studies of the golgi apparatus of pancreatic acinar cells exposed to drugs that inhibit the transport of membranes and proteins.

In a preceding article, we described alterations occurring in rat pancreas acinar cells at successive post-mortem (PM) intervals. In ultra-thin sections from samples obtained from 0.5, 1, 2, 4, 8 and 12 h, we observed in the Golgi apparatus the appearance of an anomalous membrane bound structure. Such structures are formed by tubules and vesicles that we have called tubular vesicular structure (TVS), and they are frequently located in the position corresponding to the 4th cisterna of the Golgian cisternal pile. Lobules of rat pancreas, incubated in vitro with metabolic inhibitors (such as antimycin A, sodium fluoride, sodium azide and potassium cyanide), were processed in order to be compared with the PM samples of the rat acinar cells. In sliced pieces of lobules, acid phosphatase (AcPase) and tiaminopirophosphatase (TPPase) activity were evaluated. Except for the potassium cyanide treatment, we frequently observed the TVS located at the position corresponding to the 4th cisternae (similar to those observed in the PM acinar cells). These TVS's are predominantly TPPase positive. Based on this result and the fact that the TVS's are surrounded by a membrane (as confirmed by the freeze-fracture replica results) with no structural elements inside, they seem not to correspond to autophagosomes. The TVS's, observed either at PM consecutive times or incubated with metabolic inhibitors, seem to be structures formed in response to ATP deprivation. In 0,5 h PM cells and in cells incubated for 30 and 60 min with metabolic inhibitors, the subcellular structures reacted for AcPase in the rigid lamellae, CV and lysosomes.

Autophagy-related vacuoles in mouse gallbladder epithelium.

The mouse gallbladder epithelial cells contain very heterogeneous vacuolar population. In an attempt to classify these vacuoles we identified NADPase and TPPase activity as well as the location of HRP which is used as the endocytotic marker. The results of the present study show that the vacuoles can be classified into three categories: (1) the vacuoles predominantly containing loose membrane coils related to the nascent autophagic vacuoles, (2) vacuoles containing densely packed membranes and exhibiting a positive HRP reaction, indicating the convergence of endocytotic and autophagic pathway, and (3) vacuoles composed of degraded membrane structures and containing the reaction product of NADPase activity, showing that the fusion of the lysosomes with the autophagosome-endosome took place. The highly developed cis, medial and trans Golgi compartments reflect the biosynthetic and endocytotic activity of the gallbladder epithelium.

Effects of brefeldin A on autophagy in cultured rat fibroblasts.

Effects of brefeldin A on cellular autophagy were studied in cultured rat fibroblasts. Brefeldin A inhibits the activation and membrane-binding properties of most ADP-ribosylation factors and causes the redistribution of Golgi proteins into the endoplasmic reticulum. Immunofluorescence and enzyme cytochemical methods revealed the disappearance of the Golgi apparatus and trans-Golgi network during the brefeldin A incubation. The volume fractions of autophagic vacuoles increased about threefold in cells treated with brefeldin A for 4 h and about sixfold in serum-deprived cells as compared with controls. When cells were first treated with brefeldin A for 1 h and were then deprived of serum and treated with brefeldin A for 3 h, the volume fraction of autophagic vacuoles increased about 4.5-fold as compared with untreated cells. The results showed that brefeldin A is unable to prevent serum deprivation-induced accumulation of autophagic vacuoles and that brefeldin A even when acting alone increases the volume fraction of autophagic vacuoles. It was concluded that an intact Golgi apparatus and trans-Golgi network are not essential for the formation of autophagic vacuoles. It seems also probable that ADP-ribosylation factors are not needed when vacuoles are formed.

Apical tubules in marginal cells of the differentiating stria vascularis.

BACKGROUND: Apical tubules (ATs) in marginal cells (MCs) of the stria vascularis appear in limited stages of differentiation of the MCs, but their origin and roles remain uncertain. The present study was designed to solve the problem of whether the ATs are intracellular compartments derived from the Golgi apparatus (GA). METHODS: The cochleae of Wistar rats at ages of postnatal days 1, 3, and 5 were prepared for electron microscopy and cytochemistry using thiamine pyrophosphatase (TPPase) and coenzyme A phosphatase (CoA-Pase) as marker enzymes of trans Golgi cisterns and fluorescent labelled lectin, griffonia simplicifolia agglutinin-I (GS-1). RESULTS: The ATs appeared in the apical cytoplasm of the MCs between postnatal days 1 and 5. Reaction products of TPPase and CoA-Pase activities were localized in the trans-Golgi cisterns and the ATs, which were occasionally in a close apposition to the GA. The reaction was found along the apical plasma membrane of the MCs only in case of TPPase. Heavy reactions to GS-1 were seen in the supranuclear region as well as along the apical plasma membrane of the MCs. CONCLUSIONS: The present ultrastructural and cytochemical studies indicate that the ATs, which appear in the MCs at limited perinatal stages, originate from the trans-Golgi cisterns. These ATs may be involved in the apical plasma membrane supply for the differentiation of the MCs prior to the generation of EP.

Ultrastructural localization of activity of phosphatases by low temperature incubation of unfixed cryostat sections.

In the present study, we demonstrate the activity of several phosphatases ultrastructurally in long-term (up to 24 months) cold-stored (-80 degrees C) rat tissues. Phosphatase activity was histochemically studied with the use of unfixed cryostat sections in combination with low temperature (4 degrees C) incubation conditions in order to prevent inactivation of enzyme activity and to limit the loss of ultrastructure. 5'-Nucleotidase activity was observed at plasma membranes, mainly at bile canalicular membranes of hepatocytes in liver. Thiamine pyrophosphatase activity was detected not only in trans side cisternae but also in medial and cis side cisternae of Golgi complexes in the parotid gland. Glucose-6-phosphatase activity was localized in endoplasmic reticulum as well as at the outer membrane of the nuclear envelope. Acid phosphatase reaction product was found in lysosomes. Furthermore, the localization patterns of 5'-nucleotidase and thiamine pyrophosphatase activity were compared with those obtained after different fixation procedures such as immediate chemical fixation of tissues or fixation of tissues after freezing and thawing. The results showed similar localization patterns of these enzymes after the different pretreatments. However, with respect to the ultrastructural morphology, some damage was observed in unfixed material after incubation. It can be concluded that the procedure described here enables ultrastructural localization of activity of phosphatases in long-term cold-stored tissues. This procedure will be useful for a retrospective study on archival material when histochemical parameters are needed.

[Ultracytochemical study of activity of various phosphatases during early postmortem period in experimental animals]. / Ul'tratsitokhimicheskoe issledovanie aktivnosti nekotorykh fosfataz v rannem posmertnom periode éksperimental'nykh zhivotnykh.

The activities of acid phosphatase (AP), glucose-6-phosphatase (G-6-P), and thiamine pyrophosphatase (TPP) were assayed in the epithelial cells of the main bronchi and hepatocytes of rats within 1 to 5 hours after their death. In the postmortem period, some enzymes (AC, G-6-P) retained their activity while the others (TPP) were inhibited. Furthermore, there were unusual intracellular sites of AP reaction products.

Dose-dependent effects of chloroquine on secretory granule formation in the melanotroph.

Chloroquine diverts secretory peptides from the regulated to the constitutive secretory pathway. The exact site and mechanism of the effect are not known. We studied the effect of increasing doses of chloroquine on the morphology of cultured melanotrophs from the rat pituitary. 40 microM chloroquine for 2 h, which perturbs intracellular pH gradients in melanotrophs without affecting secretion, caused swelling of a subpopulation of immature secretory granules. 200 microM chloroquine for 2 h, which diverts secretory peptides from the regulated to the constitutive pathway in the AtT-20 cell line, caused pronounced swelling of immature secretory granules, vacuolization of the trans-Golgi region and the appearance of myeloid bodies and multivesicular bodies in the cytoplasm. Golgi stacks were retained and Golgi cisternae only slightly dilated at both chloroquine concentrations. Mature secretory granules were not affected. Cationized ferritin was internalized and transported to the trans-Golgi region in the presence of 40 microM chloroquine while 200 microM chloroquine arrested internalised ferritin in peripheral multivesicular bodies. The study shows a heterogeneous effect of lower doses of chloroquine on immature secretory granules, providing a tool for studies on the relationships between condensation, acidification and peptide processing during granule formation. Chloroquine of 200 microM caused morphological changes typical for chloroquine toxicity and arrest of endocytic traffic.

Three dimensional observation of whole mount cultured cells stained with histochemical reactions by ultrahigh voltage electron microscopy.

Thick biological specimens prepared as whole mount cultured cells stained with histochemical reactions, such as thiamine pyrophosphatase, glucose-6-phosphatase, cytochrome oxidase, acid phosphatase, DAB reactions demonstrating specific cell organelles such as Golgi apparatus, endoplasmic reticulum, mitochondria, lysosomes, peroxisomes and pinocytotic vesicles, were observed by ultrahigh voltage electron microscopy at accelerating voltages of 400-1000 kV producing stereo-pairs. As a result, those cell organelles were observed 3-dimensionally and the relative relationships between these organelles demonstrated.

The Golgi apparatus and acid phosphatase-negative cisternal portions of the trans-Golgi network: ultrastructural and cytochemical studies of secretory epithelial cells in the rat lateral prostate.

Ultrastructural and enzyme-cytochemical studies were performed on the Golgi apparatus in secretory cells of the lateral prostate of normal adult rats using serial ultra-thin sections. In the trans-Golgi area, a unique membrane complex composed of tubular portions and cisternal portions showing a rigid appearance is found. This corresponds to the GERL (Novikoff 1964) or the trans-Golgi network (Griffiths and Simons 1986). From their structural similarities, the cisternal portion found in this study is considered to be the same structure as the plate-like cisterna reported by Inoue and Kurosumi (1989). As we reported previously (Kimura and Ichihara 1985), there are at least two types of acid phosphatase (AcPase) in secretory cells of the rat lateral prostate: one is located only in the structural components involved in their secretory functions and reacts readily with naphthol AS-BI phosphate; the other is a lysosomal type, which reacts well with beta-glycerophosphate. Lysosomal AcPase activity demonstrated by Gomori's method (1952) was found in a few middle- to trans-Golgi cisternae and in lysosomes. The AcPase detectable with Gomori's method and thiamine pyrophosphatase seemed to exist in part in the same cisterna on the trans side. With Robinson and Karnovsky's method (1983) for lysosomal AcPase, however, the reaction products were found only in lysosomes that were occasionally tubular in shape. On the other hand, any activity of AcPases tested could not be detected in the cisternal portion with the rigid appearance. Thus, in secretory cells of the adult rat lateral prostate in normal condition, it is considered that the cisternal portion of GERL, or the trans-Golgi network, has no relation to the processing and/or transport of AcPases.

Ultracytochemical studies on Trichomonas hominis.

The results of ultracytochemical studies on Trichomonas hominis showed that ACPase and CMPase were mainly located in the mature face sacs of the primary lysosomes, digestive vacuoles, as well as in the parabasal body. TPPase and NADPase were found in the saccules at the mature face and the intermediate saccules of parabasal body respectively. This study revealed that T. hominis had well-developed parabasal bodies. Negative COase and catalase reactions indicated that T. hominis lacked both mitochondria and microbody. Hydrogenosome was stained well with the Ur-Pb-Cu impregnation technique.

Ultrastructural and cytochemical studies of the effects of prolactin on the lateral prostate and the seminal vesicle of the castrated guinea pig.

Administration of ovine prolactin to castrated guinea pigs for 2 weeks induced hypertrophy of secretory cells in the lateral prostate when compared with the castrated controls. This was accompanied by an apparent increase in the number of profiles of granular endoplasmic reticulum and well developed Golgi complexes with dilated cisternae. An increase in the number of low-contrast electron-dense secretory granules was observed 4 weeks after prolactin treatment. In the seminal vesicle, dilatation and degranulation of granular endoplasmic reticulum and an apparent decrease in the number of secretory granules were observed 4 weeks after prolactin administration. Following castration and 2 weeks after prolactin treatment, thiamine pyrophosphatase (TPPase)-reaction product was mainly confined to 1-2 trans cisternae of the Golgi complexes in secretory cells of the lateral prostate and the seminal vesicle. In both glands, a reduction of TPPase activity was observed 2 weeks following prolactin administration, and the reaction product was totally absent after prolonged treatment for 4 weeks. The present study has provided morphological evidence that prolactin is capable of stimulating the secretory function of the lateral prostate while exerting some inhibitory effects on the seminal vesicle of the castrated guinea pig. In both glands, TPPase activity, and hence the process of glycosylation was inhibited after prolactin administration. The results from radioimmunoassay indicated that the action of prolactin on these glands could be a direct effect and not mediated through testosterone.

Presence of Golgi remnant membranes in the cytoplasm of brefeldin A-treated cells.

Brefeldin A (BFA) rapidly blocks secretion, induces disassembly of the Golgi complex and causes a redistribution of Golgi components into the endoplasmic reticulum (ER). In addition to these effects on the exocytotic pathway, BFA has been shown to induce fusion of endosomal membranes with the trans-Golgi network in some cell types. To better understand the mechanism through which BFA disrupts the exocytotic traffic, we have examined its effects on the ultrastructural organization of the Golgi complex. Within minutes of exposure to BFA, the Golgi cisternae were fragmented into a number of small tubules and vesicles, many of which had a non-clathrin coat on their cytosolic surface. In addition, a complex structure consisting of anastomosing tubules and associated vesicles appeared in the cytoplasm of cells incubated with BFA for 10 min or longer. These tubular networks were permanent, distinct structures separated from the ER cisternae. They contained cis, middle, and trans Golgi proteins as well as the lipid analogue C5-DMB-ceramide. Furthermore, secretory proteoglycans en route through the Golgi were retained in the lumen of the tubular networks. As judged by the endocytosis of cationized ferritin, endosomes do not contribute to the formation of these tubular networks. Reassembly of the Golgi complex after BFA incubation involved fragmentation and reorganization of the tubular networks as well as fusion with vesicles budded from the ER. We conclude that although in the presence of BFA the bulk of Golgi membranes are induced to fuse with the ER, as indicated by the detection of Golgi markers in this organelle, a fraction of these membranes remain in the cytoplasm organized as Golgi remnants.

Cytochemical and stereological analysis of rat cortical astrocytes during development in primary culture. Effect of prenatal exposure to ethanol.

This study has investigated the effect of prenatal alcohol exposure on the qualitative and quantitative ultrastructure of proliferating and differentiated astrocytes in primary cultures as well as on the cytochemical activity of several subcellular phosphatase markers, including acid phosphatase, uridine diphosphatase, thiamine pyrophosphatase, 5'-nucleotidase and glucose-6-phosphatase. The astrocytes were obtained from 21-day-fetuses of both control and alcohol-fed rats. Our results show that several cell components, such as mitochondria, rough endoplasmic reticulum and lysosomes, exhibit qualitative and/or quantitative ultrastructural changes during the process of astrocyte maturation. In some cases these morphological changes are accompanied by variations in the cytochemical activity of enzymes located in these and other cell components, suggesting that these enzymes, and therefore the functional state of these organelles, are modulated during astrocyte development. When prenatally exposed to ethanol, both proliferating and differentiated astrocytes showed striking ultrastructural alterations compared with controls, including an increment of lysosomes as well as a decrease in the values of stereological parameters relative to mitochondria, rough endoplasmic reticulum and Golgi apparatus. Cytochemical analysis of these cells indicates that prenatal exposure to ethanol decreased the activities of all the enzymes tested, except for acid phosphatase, which was increased in both groups of treated astrocytes. These results suggest that prenatal exposure to ethanol could affect astrocytes during development in two different but probably complementary ways: a) by causing a delay in astrocyte maturation and, b) by inducing a direct toxic effect on these cells.

Retinol stimulates Golgi apparatus activity in cultured bovine mammary gland epithelial cells.

Biochemical and electron microscopic studies have indicated that the Golgi apparatus responds to retinol. The purpose of this investigation was to visualize and record with living cells the rapidity of the response to retinol. A rapid response of the Golgi apparatus to retinol (1.75-17.5 mumol/L) added to the culture medium was observed using video-enhanced light microscopy with bovine mammary epithelial cells. The response was manifested within 1 min as a marked movement of membranes within the Golgi apparatus zone. In subsequent electron microscope preparations of the cells, only minor changes were observed and were restricted to increased numbers of normal-appearing membranes and vesicles associated with the trans Golgi apparatus face of the retinol-treated cells.

Structural and cytochemical study of the rat exocrine pancreas treated with dl-ethionine. I. Multilayered bodies and lesioned areas.

The fine structural changes and the reactivity for acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) were studied in thin sections from rat pancreatic acinar cells exposed to dl-ethionine for 2-10 days. The cells from ad libitum and pair-fed controls exhibit occasionally 0.2-0.6 microns circular profiles showing reaction for AcPase and considered as presumptive lysosomes. At days 2 and 4 of dl-ethionine treatment the acinar cells exhibit presumptive lysosomes, autophagosomes and membrane-bounded cytoplasmic areas devoid of electron density and AcPase activity, containing scattered membranous elements. These regions were named lesioned areas. On 6th, 8th and 10th days a membrane bound anomalous cytoplasmic structure that represents a dense pile of layered membrane-like material (multilayered bodies, MB) was seen. The MBs consistently show AcPase activity and in rare instances TPPase activity. Freeze fracture studies reveal that the limiting membrane of the MBs has intramembranous particles whereas the multilayered membranous contents are devoid of such particles. The structure and disposition of the lamellae of the MBs seen in the replicas are similar to those of artificially prepared phospholipidic membranes.

Cytochemical localization of enzyme markers in Tritrichomonas foetus.

Cytochemical techniques associated with transmission electron microscopy were used for the localization in Tritrichomonas foetus of enzymes used as markers of different cell structures. Reaction product indicating the presence of Mg(2+)-adenosine triphosphatase (Mg(2+)-ATPase) and 5'-nucleotidase was observed in the plasma membrane. Glucose-6-phosphatase was seen in association with the endoplasmic reticulum, revealing its organization as parallel cisternae. Thiamino-pyrophosphatase was located in the cis-most region of the Golgi complex. Acid phosphatase was found within lysosomes as well as in several cisternae of the Golgi complex, in contrast to previous observations in mammalian cells. These observations provide support for the use of enzyme markers in future studies on cell fractionation of T. foetus.

Lysosomal depletion in macrophages from spleen and foot lesions of Leishmania-infected hamster.

Analysis of lysosomes through acid phosphatase cytochemistry at the electron microscopy level has been performed in spleen and foot lesions from Leishmania-infected hamsters. The results showed that there is lysosomal depletion in macrophages from Leishmania donovani chagasi-infected hamster spleen and similar findings were obtained from foot lesions of Leishmania mexicana amazonensis-infected hamsters. The distribution of acid phosphatase and thiamine pyrophosphatase was also examined in the Golgi apparatus. It was possible to demonstrate that the activity of ACP is absent in infected macrophages from spleen and foot lesions of Leishmania-infected hamster while the distribution of TPP was very similar in control and infected macrophages from both systems. These results provide evidence that the lysosomal depletion can occur at the ACP synthesis and/or glycosylation level.

Ultrastructural localization of phosphatase activity in the guinea pig pineal gland by the cerium technique.

Thiamine pyrophosphatase (TPPase), nucleoside diphosphatase (NDPase), and glucose-6-phosphatase (G-6-Pase) were localized by the cerium technique in guinea pig pinealocytes and compared with the corresponding lead technique. NDPase and TPPase were also compared at different pH values using the cerium technique. Vibratome sections of perfusion-fixed tissue were incubated with cerium chloride or lead nitrate. Substrates used were thiamine pyrophosphate (for TPPase), sodium inosine diphosphate (NDPase), and disodium glucose-6-phosphate (G-6-Pase). The 1-2 trans saccules of the Golgi apparatus showed TPPase and NDPase activity but none for G-6-Pase. The endoplasmic reticulum (ER) cisternae and perinuclear space had NDPase and G-6-Pase activity but not TPPase. The abluminal plasmalemma of endothelial cells and the plasmalemma of Schwann cells demonstrated TPPase and NDPase activity but the luminal plasmalemma of the endothelial cells and the plasmalemma of pinealocyte processes showed only NDPase activity. TPPase was active at all pH values tested, but NDPase was most active at pH values of 6.5 and 7.0. Lead phosphate precipitate was frequently seen in nuclei, perinuclear space, ER cisternae, and "synaptic" vesicles when lead was used as the capturing agent. These sites were usually not labeled when cerium was used.

The occurrence of the Golgi apparatus in mouse oocytes. Demonstration of thiamine pyrophosphatase activity.

Location of thiamine pyrophosphatase activity as a marker of the Golgi apparatus was studied ultracytochemically in mouse oocytes with germinal vesicle (OGV), oocytes at metaphase I (OMI) and oocytes at metaphase II (OMII), and further in cells of the respective cumulus oophorus serving as comparative objects. TPPase activity in cumulus oophorus cells and in OGV was found exclusively in the Golgi apparatus. In OMI the reaction product of TPPase activity was observed in isolated smooth vesicles, and in only one case in structures identifiable as the Golgi apparatus. In OMII the occurrence of TPPase activity was also recorded in isolated smooth vesicles in cortical cytoplasm and further, exceptionally, in smooth concentrically arranged vesicles or tubules. The TPPase activity was not present in vesicular complexes. The results have shown that after the resumption of meiosis the occurrence of the reaction product of TPPase activity drops abruptly due to the reduction of the Golgi apparatus. Changes affecting the Golgi apparatus after the resumption of meiosis are related to the loss of the nucleus after the germinal vesicle breakdown.