Cytochemical Studies Research Articles

ILA 147 immunoreactivity of the bull spermatozoa membrane during epididymal maturation

A cytochemical quantitative study was carried out to detect immunostaining of bull spermatozoa during epididymal maturation using an ILA 147 monoclonal antibody and a standard immunoperoxidase method. This antibody recognizes a bovine panleukocyte determinant. Microdensitometric measurements were made on spermatozoa collected from different sites of the male genital tract (caput, corpus and cauda epididymidis and ductus deferens). On the basis of ILA 147 staining, at least 5 subpopulations of sperm cells from each site of the genital tract were found. These subpopulations showed: 1) immunostaining in the acrosome domain and in the cytoplasmic droplet in a proximal position; 2) immunostaining in the acrosome domain and in the distal cytoplasmic droplet; 3) immunostaining in the acrosomal region and lack of a cytoplasmic droplet; 4) immunostaining only in the cytoplasmic droplet; 5) lack of a cytoplasmic droplet and absence of staining of the head. The possible relationship between the presence of ILA 147 on the sperm head and the maturation process was evaluated, and we suggest that the most significant changes in ILA 147 expression occur in the corpus epididymidis. The absence of immunocytochemical staining in some spermatozoa may be related to plasma membrane damage due to spontaneous peroxidative damage of lipids.

Pregnancy alters glucose-6-phosphate dehydrogenase trafficking, cell metabolism, and oxidant release of maternal neutrophils.

Pregnancy is associated with changes in host susceptibility to infections and inflammatory disease. We hypothesize that metabolic enzyme trafficking affects maternal neutrophil activation. Specifically, immunofluorescence microscopy has shown that glucose-6-phosphate dehydrogenase (G-6-PDase), the rate-controlling step of the hexose monophosphate shunt (HMS), is located near the cell periphery in control neutrophils but is found near the microtubule-organizing centers in cells from pregnant women. Cytochemical studies confirmed that the distribution of the G-6-PDase antigen is coincident with functional G-6-PDase activity. Metabolic oscillations within activated pregnancy neutrophils are higher in amplitude, though lower in frequency, than activated control neutrophils, suggesting limited HMS activity. Analysis of radioisotope-labeled carbon flux from glucose to CO(2) indicates that the HMS is intact in leukocytes from pregnant women, but its level is not enhanced by cell stimulation. Using extracellular fluorescent markers, activated pregnancy neutrophils were found to release reactive oxygen metabolites (ROMs) at a lower rate than activated control neutrophils. However, basal levels of ROM production in polarized pregnancy neutrophils were greater than in control neutrophils. Microtubule-disrupting agents reversed the observed changes in G-6-PDase trafficking, metabolic oscillations, and ROM production by maternal neutrophils. G-6-PDase trafficking appears to be one mechanism regulating ROM production by maternal neutrophils.

Lectin-histochemical and -cytochemical study of periodic acid Schiff-positive lysosome granules as a histological feature of the female mouse kidney.

Renal proximal straight tubules (PST) of the female mouse contain periodic acid Schiff-positive lysosome granules. An excellent example of this is found in the kidneys of female DBA/2Cr mice. In the present study, lectin-histochemistry showed that lectin-positive granules occur in the PST of DBA/2Cr mice. Out of twenty-one lectins studied, the granules bound WGA, s-WGA, LEL, STL, DSL, GSL-II, VVL, RCA-I, ECL, PSA, LCA and PHA-E. Such granules were also observed in the proximal convoluted tubules (PCT). In addition, heterogeneous binding to the SBA or DBA was observed in the PST. Lectin-cytochemistry for s-WGA, STL, VVL, RCA-I, ECL and PSA, showed that: 1) lysosomes bind a higher level of s-WGA or STL than VVL, RCA-I, ECL or PSA; 2) PSA binding is similar in PST and PCT; 3) there are many PCT lysosomes that are negative for s-WGA, STL, VVL, RCA-I, and ECL lectin binding; and 4) s-WGA binding is highly specific to the lysosomes of the PST. Based on the binding specificities of each lectin, it was suggested that the mannose content of PST and PCT lysosomes is similar, and that PST lysosomes have a high level of N-acetylglucosamine, N-acetylgalactosamine, galactose or galactosyl (beta 1, 4) N-acetylglucosamine.

Degeneration of retinal neuronal processes and pigment epithelium in the early stage of the streptozotocin-diabetic rats.

Early pathological and electro-physiological changes of the retina in the streptozotocin (STZ)-diabetic rats were investigated through optical and electron microscopy in two strains and electro-retinography in one strain. In Sprague-Dawley (SD) rats I month after the onset of diabetes, the thickness of the inner plexiform layer (IPL) and photoreceptor segment layer (PSL) was significantly reduced by 9.9% and 18.9%, respectively (P < 0.01, P < 0.05). In Brown-Norway (BN) rats STZ-diabetic for 1 month, the thickness of the IPL was also significantly reduced by 15.7% (P < 0.05). Cytochemical study using peanut agglutinin (PNA), a lectin binding selectively to the cone photoreceptor-associated domains of the inter-photoreceptor matrix, revealed a marked reduction in intensity, number and length of the PNA-binding cone photoreceptors. Electron microscopy showed deepened hollows in the basal infoldings of the retinal pigment epithelium (RPE) of STZ-rats diabetic for 1 month and large concavities into the cytoplasm in STZ-rats diabetic for 6 months. Blood vessels in the retina and choroid were unremarkable. Single-flash electro-retinogram revealed a reduction in the amplitudes of alpha- and beta-waves of electro-retinogram (ERG) of 1 month STZ BN rats (P < 0.05). These findings indicate that the degeneration of rods/cones in the PSL and RPE are the most prominent pathological alteration sites in the early stage of diabetic rats.

Polyphosphate in Zygomycetes: a cytochemical study

The inorganic phosphorus content, and distribution, structure and localization of polyphosphate in mycelia of zygomycetous fungi was evaluated. Ultrastructural cytochemistry was successfully used to identifying the localization and distribution of polyphosphate in Absidia cylindrospora, Gongronella butleri and Mucor javanicus. The results revealed differences in the cytochemical staining pattern in all species studied and a uniform labeling on the cellular surface (cell wall and citoplasmic membrane). Reaction products were observed in intracellular structures and cellular membrane. Intracellular staining was observed in trabecullar, vacuolar and vesicular structures, in dense bodies and in the cytoplasm. However, the cytochemical staining intensity varied during the cellular growth. Analytical procedure revealed the phosphorus content during cell growth. The results demonstrated that phosphorus content varied during cultivation time and could be related to the polyphosphate cytochemical staining. Polyhosphate functions such as storage and utilization are discussed.

An epidemiologic study to screen for chronic myelocytic leukemia in war victims exposed to mustard gas.

Chemical agents such as mustard gas (or sulfur mustard), which has alkylating characteristics, were used against Iranian combatants in the Iraq-Iran war. Previous studies have not shown a strong link between these chemical agents and the development of chronic myelocytic leukemia (CML). The purpose of this study was to evaluate the increased risk of CML development in Iranian soldiers exposed to mustard gas during the war. Based on a descriptive study of 2,500 cases with documented exposure to various chemical warfare agents, 665 patients had documented exposure to mustard gas. We screened the latter using the leukocyte alkaline phosphatase (LAP) test and performed further cytochemical studies on cases with positive results. From among the 665 cases with documented exposure to mustard gas, 9 cases had LAP scores < 20; 2 of these 9 cases had CML and a score of zero (0.3%). We detected cytogenetic abnormalities in 7 patients with low LAP scores and atypical lymphocytes of 5-11% in 40 patients. The risk ratio of CML developing in victims exposed to mustard gas (cutaneous or respiratory) may be higher in comparison with the normal population, although confounding factors (e.g., the possibility of exposure to combined chemical agents, excluding patients who did not manifest blisters) limited our results. Because the increased development of CML in young patients with a documented history of exposure to mustard gas cannot be disregarded, further studies are needed.

Localization and functional role of hepatocyte growth factor (HGF) and its receptor c-met in the rat developing cerebral cortex

Development of the cerebral cortex is a series of precisely timed proliferative, migratory, and maturational processes. Hepatocyte growth factor (HGF) is a pleiotrophic cytokine, which plays important roles in the organogenesis and regeneration of various tissues, both during development and in the adult, due to its mitogenic, motogenic and morphogenic activities. In the present study, we examined expression and functional roles of HGF and c-Met during development of the rat cerebral cortex. Quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) revealed that expression levels of c- met and HGF mRNAs were increased in the cerebral cortex during late embryonic development and peaked at E18. Immunohistochemical analyses revealed that c-Met-immunoreactivity (IR) was localized to the preplate (PP), with weaker-IR in neuroepithelial layer (NE) at embryonic day 14 (E14). At E16, c-Met-IR was present in the cortical plate (CP) and the intermediate zone (IZ), with a weak presence in the ventricular zone (VZ). On the other hand, HGF-IR was present in NE and VZ at E14 and E16, respectively. HGF-IR appeared in cortical plate tissue from E16 onward. Double labeling immunofluorescent cytochemical studies revealed that c-Met-IR was localized both in TuJ-1-IR- and non-TuJ-1-IR-cells, purified from E18 cerebral cortex in vitro, suggesting the presence of c-Met-IR in postmitotic neurons as well as in neuroepithelial cells. c-Met-IR was strong in cell bodies and neurites shortly after in vitro culture, while at 7DIV c-Met-IR decreased in neurites and was evident in growth cones. HGF dose dependently supported neuronal survival in vitro under serum-deprived conditions. In a transwell culture chamber, HGF increased neuronal migration, and co-incubation with functional blocking antibody against HGF abrogated this motogenic effect of HGF. These lines of evidence suggest that HGF is involved in the development and maintenance of cortical neurons during differentiation, motogenesis, neuritogenesis and neuronal survival.

Second Messengers in the Locomotor-Sensory System of First Multicellulars. The IP 3 -Containing Compartments in Chemoreceptor Cells of the Comb Jelly Beroe cucumis from Data of Cytochemical Study

Result of the performed electron microscopy cytochemical study was an indirect detection of intracellular localization of inositol 1,4,5-triphosphate according to sites of its binding with specific phosphatase in receptor and secretory cells of epithelium surrounding actinostome of the comb jelly Beroe cucumis. The highest level of the deposit of the enzymatic cytochemical reaction product was revealed in chemoreceptor cells of I type. In all cells of the epithelium as well as in nerve terminals and neuroplexus elements, the following compartments had the highest deposit content: cellular membrane and glycocalyx areas, microtubules, microfilaments, mitochondria, neurotubules, and structures of the synaptic apparatus. The obtained data expand the picture of cytochemical distribution and of the contents of the deposit compatible with localization of components of inositol pathway in receptor cells of the locomotor-sensory system of Beroe cucumis [1, 2] and indicate its essential role in the mechanism of chemosensory transduction.

Localization of phosphatase responsible for hydrolysis of inositol-1,4,5-triphosphate in the olfactory lining of sturgeons

The paper presents results of a cytochemical study of localization of phosphatase responsible for hydrolysis of inositol 1,4,5-triphosphate (ITP) in the olfactory lining of true sturgeons (the sturgeon, starred sturgeon, and sterlet). Reaction products as a dark discrete granules are localized in the apical parts of epithelium, practically in the same manner in all the species studied. The precipitate is found on the plasma membranes of cilia, microvilli, and clava of the olfactory cells. Occasionally, the precipitate is also found in the cilia, basal bodies, and rootlets of microvillar cells. The ITP-hydrolyzing phosphatase is supposed to restrict development of transduction process by removing excess messengers from the operating system. The data obtained indicate that in the true sturgeons, the phospholipase cascade of olfactory transduction is concentrated predominantly in the cilia and microvilli of olfactory cells.

In situ heparin-induced peroxisomal reticulum and biogenesis of peroxisomes in pulmonary intravascular macrophages (PIMs) of caprine lung: an ultrastructural and cytochemical study.

Pulmonary intravascular macrophages (PIMs) contain a unique electron-dense globular surface-coat which is sensitive to heparin treatment, halothane anesthesia, and the digestive effect of lipolytic lipase (LPL), suggesting that the coat is predominantly composed of lipoproteins. In the present study, evidence is presented that heparin, when administered intravenously in goats, potentiated both the translocation of the surface-coat into the vacuolar system and the expansion of the Golgi apparatus. Sequentially, these changes were followed by proliferation of peroxisomes in combination with peroxisomal reticulum (PR), a transient precursor of this organelle. The peroxisomes, as well as PR, reacted positively for catalase after aldehyde fixation and 3,3'-diaminobenzidine (DAB) staining. In addition to their role as phagocytes, the ultrastructural and cytochemical detection of peroxisomes suggests a functional capacity of the PIMs, which may be adaptable to the circulating level of free fatty acids (FAAs).

Ultrastructural and cytochemical features of the supramedullary neurons of the pufferfish Diodon holacanthus (L.) (Osteichthyes)

Exceptionally high DNA contents were found in supramedullary neuron (SN) nuclei of the pufferfishDiodon holacanthus by quantitative microfluorimetric assay. This phenomenon has been explained by endoreplication, the functional significance of which is still unclear. In this view, the peptidergic nature and large dimensions make the teleostean clustered SN an interesting model for investigating the relationships between endoreplication, nuclear morphology and biosynthetic cellular activity.In this paper, we present a cytochemical and ultrastructural study on the SN of D. holacanthus (Tetraodontiformes). The nucleolar and nucleus structures suggest an intense production of ribosomal components in order to satisfy high cellular demands for protein synthesis. Accordingly, the cytoplasmic compartment presents an extensive rough endoplasmic reticulum, well-developed Golgi apparatus and a remarkable vesicular traffic. These features suggest that SN are engaged in an intense process of protein biosynthesis.The SN are completely surrounded by processes of different types of glial cells. The glial cells may be considered part of the SN cluster.

Cytochemical study of the involvement of cell organelles in formation and accumulation of fibrillar amyloid in the pancreas of NORbeta transgenic mice.

Phosphatase ultrastructural cytochemistry was used to evaluate the participation of cytoplasmic organelles in the accumulation of fibrillar amyloid beta (Abeta) in exocrine acinar cells and in macrophages of the pancreas of transgenic mice overexpressing a carboxy-terminal fragment of Abeta protein precursor (ABPP). Nucleoside diphosphatase (NDPase) and glucose-6-phosphatase (G6Pase) were used as cytochemical markers of the endoplasmic reticulum (ER), thiamine pyrophosphatase (TPPase) as a marker of the Golgi apparatus (GA), and acid phosphatase (AcPase) as a marker of lysosomes. Monoclonal antibody 4G8 raised against the 17-24 aa sequence of human Abeta protein was used for immunogold localization of fibrillar Abeta. The results of this study indicate that the formation of Abeta in acinar cells occurs directly in the vacuolar areas of the rough ER (RER) without evident participation of the elements of the GA, whereas an intimate structural relation with primary lysosomes suggests their role in modification or digestion of the deposited amyloid. In macrophages, fibrillar amyloid was present in numerous cytoplasmic vacuoles located frequently in close proximity to flattened saccules of the ER. This structural pattern revealed similarity to that observed previously in microglial cells producing fibrillar PrP amyloid in scrapie-infected mice and Abeta in brains of human elderly patients and in Alzheimer's type brain pathology.

Succinic dehydrogenase activity in human muscle mitochondria during aging: a quantitative cytochemical investigation

A quantitative cytochemical study has been carried out on succinic dehydrogenase (SDH) activity in biopsy samples of vastus lateralis (VL) and anterior tibialis (AT) muscles from healthy men undergoing orthopaedic surgery. According to their age, the patients were divided into: young (25.0±4.4 years), middle-aged (50.4±7.5 years) and old (75.5±3.9 years) groups. Bioptically excised samples were processed for copper ferrocyanide preferential SDH cytochemistry. By a computer-assisted image analyser, we calculated the ratio ( R): overall area of the precipitates due to the enzyme activity/area of each mitochondrion. No significant difference was found among the three age groups, despite an 8% increase of R in the adult vs. the other groups. R values are related to mitochondrial morphofunctional features since they may be modulated by enzyme activity and the physico-chemical conditions of the organelle membranes. Thus, R quantitation enables to estimate the mitochondrial capacities for adenosinetriphosphate provision. In this context, our present findings confirm previous data reporting a substantial age-related stability of muscle mitochondrial enzyme levels. In aging, energy-deficient sarcomeres are supported to be negatively selected and eliminated, while the surviving ones appear to maintain an adequate SDH activity.

The shell glands in some calanoid copepods (Crustacea)

This study represents the first structural, ultrastructural, and biochemical investigation of the shell glands in calanoid copepods. These glands, located inside the points of the last prosomal segment, constitute voluminous syncytial secretory units, each of which extends into an excretory canalicule with a cellular or syncytial wall. The canalicules merge into two collector canals, or shell ducts, that rejoin the oviducts and then open into the egg-laying ducts. Each secretory unit synthesizes heterogeneous granules containing both light and predominantly dense material. Exocytosis of these mature secretory granules occurs in an apical excretory chamber. In freshwater species, the modifications observed during the secretory cycle emphasize a gradual discharge from the secretory units in the hours following laying. Cytochemical and biochemical studies of the secretions reveal the presence of N-acetylglucosamine, galactose, and N-acetylgalactosamine glycoconjugated proteins.

Ultracytochemical study of medullary bone calcification in estrogen injected male Japanese quail.

Fine structural and cytochemical studies were performed to clarify the pattern of medullary bone calcification, specifically in relation to sulfated glycosaminoglycans, by using estrogen-induced medullary bone of male Japanese quails. Tibiae were collected at 4 and 7 days after estrogen treatment. Medullary bone had developed inward toward the marrow cavity, and calcification had begun near the cortical bone and deeper parts of the trabeculae, accompanied by wide osteoid at extending tips and surface areas of the trabeculae. Sulfated glycosaminoglycans, detected by high iron diamine (HID), were distributed in the matrix in a pattern similar to that of calcified matrix of the trabeculae. Cortical bone was negatively stained by HID. In undecalcified specimens, calcified nodules were seen in areas undergoing calcification. Globular structures composed of fine filamentous materials, a marginal dense layer, and central core, were also observed in the matrix of decalcified specimens. Both the calcified nodules and globular structures showed the same distribution pattern, i.e., they were dispersed at surface areas and coalesced in the deeper areas of the matrix. The globular structures were exclusively positive for HID-thiocarbohydrazide-silver protein (HID-TCH-SP) stain, indicating the localization of sulfated glycosaminoglycans. These results strongly suggest that medullary bone calcification progresses by the coalescence of calcified nodules and that sulfated glycosaminoglycans play an important role for the regulation of globular calcification.

Nuclear changes and acrosome formation during spermiogenesis in Euchistus heros (Hemiptera: Pentatomidae)

Ultrastructural and cytochemical studies were carried out on nuclear changes and acrosome formation during the spermiogenesis of the phytophagous bug Euchistus heros. The development of the nucleus involves changes in the shape and in degree of chromatin condensation: initially it is dispersed and with a low-electron density, then assumes a fibrillar arrangement and finally compacts in an electron-dense material. The acrosome is formed by the Golgi complex and presents unusual morphological features during its development. The reaction product of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase activities were detected during various stagesof acrosome development. In contrast, residues of α-N-acetylgalactosamine and basic proteinswere only reported in the intermediate and late stages of the differentiation process, respectively.

Life without a cell membrane: regeneration of protoplasts from disintegrated cells of the marine green alga Bryopsis plumosa.

When the multi-nucleate giant cells of the green alga Bryopsis plumosa (Huds.) Ag. are injured, the protoplasm is extruded from the cells and can generate spontaneously numerous new cells. The cell organelles aggregate rapidly in seawater and become covered with a gelatinous envelope within 15 minutes. A lipid cell membrane is formed inside the envelope within 9 to 12 hours and about 15% of the original cell membrane is recycled to make the membrane of new protoplasts. Cytochemical studies using Nile Red and various enzymes revealed that the primary envelope is initially composed of polysaccharides, and then transformed into a polysaccharide-lipid complex. Fluorescein diacetate staining showed that the primary envelope has some characteristics of a cell membrane including semi-permeability and selective transport of materials. The aggregation of cell organelles appears to be mediated by two kinds of materials, one present in vacuolar sap and the other on the surface of the cell organelles. About a thousand new cells were generated from a single disintegrated branch and 40% of them eventually developed into mature plants.

Compartmentation of photosynthesis in cells and tissues of C4 plants

Critical to defining photosynthesis in C(4) plants is understanding the intercellular and intracellular compartmentation of enzymes between mesophyll and bundle sheath cells in the leaf. This includes enzymes of the C(4) cycle (including three subtypes), the C(3) pathway and photorespiration. The current state of knowledge of this compartmentation is a consequence of the development and application of different techniques over the past three decades. Initial studies led to some alternative hypotheses on the mechanism of C(4) photosynthesis, and some controversy over the compartmentation of enzymes. The development of methods for separating mesophyll and bundle sheath cells provided convincing evidence on intercellular compartmentation of the key components of the C(4) pathway. Studies on the intracellular compartmentation of enzymes between organelles and the cytosol were facilitated by the isolation of mesophyll and bundle sheath protoplasts, which can be fractionated gently while maintaining organelle integrity. Now, the ability to determine localization of photosynthetic enzymes conclusively, through in situ immunolocalization by confocal light microscopy and transmission electron microscopy, is providing further insight into the mechanism of C(4) photosynthesis and its evolution. Currently, immunological, ultrastructural and cytochemical studies are revealing relationships between anatomical arrangements and photosynthetic mechanisms which are probably related to environmental factors associated with evolution of these plants. This includes interesting variations in the C(4) syndrome in leaves and cotyledons of species in the tribe Salsoleae of the family Chenopodiaceae, in relation to evolution and ecology. Thus, analysis of structure-function relationships using modern techniques is a very powerful approach to understanding evolution and regulation of the photosynthetic carbon reduction mechanisms.

The vesiculo-vacuolar organelle (VVO). A new endothelial cell permeability organelle.

A newly defined endothelial cell permeability structure, termed the vesiculo-vacuolar organelle (VVO), has been identified in the microvasculature that accompanies tumors, in venules associated with allergic inflammation, and in the endothelia of normal venules. This organelle provides the major route of extravasation of macromolecules at sites of increased vascular permeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), serotonin, and histamine in animal models. Continuity of these large sessile structures between the vascular lumen and the extracellular space has been demonstrated in kinetic studies with ultrastructural electron-dense tracers, by direct observation of tilted electron micrographs, and by ultrathin serial sections with three-dimensional computer reconstructions. Ultrastructural enzyme-affinity cytochemical and immunocytochemical studies have identified histamine and VPF/VEGF bound to VVOs in vivo in animal models in which these mediators of permeability are released from mast cells and tumor cells, respectively. The high-affinity receptor for VPF/VEGF, VEGFR-2, was localized to VVOs and their substructural components by pre-embedding ultrastructural immunonanogold and immunoperoxidase techniques. Similar methods were used to localize caveolin and vesicle-associated membrane protein (VAMP) to VVOs and caveolae, indicating a possible commonality of formation and function of VVOs to caveolae.

Pyriform cell differentiation in Podarcis sicula is accompanied by the appearance of surface glycoproteins bearing alpha-galNAc terminated chains.

The present histochemical and cytochemical study using a lectin panel (WGA, GSI-A4, GSI-B4, PSA UEA-I, PNA, LCA, Con-A, DBA, MPA, BPA) has demonstrated that, in Podarcis sicula, the differentiation of small follicle cells into pyriform cells by means of intermediate cells is accompanied by the appearance of glycoproteins bearing alpha-GalNAc terminated O-linked side chains on the cell surface. The distribution of DBA- and MPA-binding sites over the follicular epithelium changed during the different stages of oocyte growth. DBA- and MPA-binding sites first appeared at the beginning of folliculogenesis within the zona pellucida (ZP) and on the surface of small cells, i.e., the stem cells of pyriform cells. Afterward, labeling was evident on the cell surfaces of intermediate cells and, later on, also of pyriform cells. On the other hand, no labeling was detected on the small cells located under the basal lamina, which, reportedly, do not differentiate into pyriform cells (Filosa et al. J. Embryol. Exp. Morphol., 1979; 15:297-316). Once pyriform cells were differentiated, the distribution of DBA- and MPA-binding sites over the follicular epithelium remained unchanged until intermediate and pyriform cells underwent apoptosis (Motta et al. J. Exp. Zool., 1996; 276:233-241) and the follicular epithelium transformed into a monolayer composed of small follicle cells only (Filosa Mon. Zool. Ital., 1973; 7:151-165). During this stage of oocyte growth, DBA and MPA labeling gradually decreased to completely disappear in the follicular epithelium of vitellogenic follicles. It is noteworthy that the observed changes in the distribution of DBA- and MPA-binding sites represent the first evidence recognized by lectins of a gradual modification of surface glycoprotein distribution over the follicular epithelium in the ovarian follicles of nonmammalian vertebrates so far studied. Finally, the zona pellucida (ZP), characterized by the presence of GalNAc, GluNAc, Man, and Gal, was demonstrated to be first synthetized by the oocyte and later on by the follicle cells.