William J. Dougherty

Meiotic chromosome complements and nuclear DNA contents of four species of shrimps of the genus Penaeus

Meiotic chromosome spreads were prepared from testicular lobes of 4 species of penaeid shrimps to determine numbers of chromosomes and meiotic phases. DNA content of nuclei isolated from shrimp hemocytes of these species was estimated by flow cytometry. Meiotic chromosome spreads indicated that the haploid chromosome number of Penaeus aztecus, P. duorarum, and P. vannamei was 44, while that ofP. setiferus was 45. These counts are consistent with diploid chromosome numbers reported previously for each of these species. No trivalents or quadrivalents were found in meiotic prophase. These 4 species had approximately the same genome size (approximately 70% of the human genome) as measured by flow cytometry. It is suggested that Robertsonian chromosome rearrangement has accompanied the speciation process in the genus Penaeus without polyploidization. Chromosome research in the decapod Crustacea has advanced slowly (Farmer, 1974; Milligan, 1976; Hughes, 1982; Hayashi and Fujiwara, 1988). Milligan (1976) and Hayashi and Fujiwara (1988) provided new techniques to obtain mitotic chromosome metaphase preparations in penaeid shrimps, but these works are still concerned only with chromosome counts and comparison of chromosome numbers between species. Thus, no karyotype has ever been described in this animal group. Although the small size and large numbers of chromosomes present some difficulty, determination of the genome size and examination of meiotic chromosome phase may provide additional information. In this paper, we report observations on meiotic chromosome phases in 4 species of shrimps of the genus Penaeus, with determinations of their genome size.

Effect of denervation on 'synaptic' ribbon populations in the rat pineal gland

SummaryThe formation of pineal ‘synaptic’ ribbons (SR) may be directly related to the adrenergic innervation of the gland. In order to clarify this relationship, SR populations at various times from 12 h to 14 days after pineal denervation were morphometrically analysed by electron microscopy. Pineal denervation was accomplished by bilateral superior cervical ganglionectomy. A decrease in nocturnal pineal SR numbers, indicating a reduction in SR formation, was demonstrated 12 to 24 h after pineal denervation. Seventy-two hours after ganglionectomy SR numbers were comparable with those in nocturnal intact and sham-operated controls. Thereafter, 7 and 14 days after ganglionectomy, SR numbers exceeded nocturnal intact and sham-operated controls. Administration of isoproterenol, a beta-adrenergic receptor agonist, 24 h after denervation significantly increased SR numbers over those in untreated rats denervated 24 h earlier. Thus SR formation remained responsive to adrenergic receptor stimulation in the absence of an intact adrenergic innervation. Further, the increase in SR numbers following subacute (7 to 14 days) denervation indicated that SR formation was not dependent on an intact innervation or the presence of endogenous (pineal) norepinephrine. On the basis of these results, we suggest that SR formation may be related structurally as well as functionally to adrenergic receptors on the rat pinealocyte.

Electron microscopical and histochemical observations on melanized sperm and spermatophores of pond-cultured shrimp, Penaeus vannamei

Black spermatophores were collected by manual ejaculation of pond-cultured Penaeus vannamei males and were prepared for light and electron microscopical study. Light microscopy revealed varying numbers of individual and aggregated yellow-brown to black polymorphic pigment droplets measuring 2 to 400 μm in diameter in spermatophore capsules. Some black spermatophores contained mostly degenerate sperm, while others contained mixtures of normal-appearing unistellate sperm, abnormal spikeless sperm, and degenerating sperm. Yellow-brown to black oval pigment droplets measuring 1 to 2.5 μm in diameter were encountered in the nuclei both of normal-appearing unistellate sperm and of abnormal spikeless sperm. Degenerating sperm exhibited no spikes and no pigment droplets in the remains of the sperm cell bodies. Oval pigment droplets occurred also among sperm in the extracellular matrix of spermatophores. Capsular, matrical, and intranuclear pigment droplets all gave histochemical reactions characteristic of melanin; i.e., pigment droplets were bleached by acidified permanganate, gave positive Schmorl and methenamine silver reactions, and adsorbed ferrous ions. Acid phosphatase activity was not detected in the sperm or spermatophores. Electron microscopy revealed oval, electron-dense droplets within sperm nuclei of both unistellate and spikeless sperm. These droplets corresponded in size to the pigment droplets seen in sperm by light microscopy. Intranuclear droplets were bounded by a single membrane, exhibited fairly homogeneous internal contents, and were frequently enmeshed in strands of intranuclear chromatin threads. Spikeless sperm, in addition to containing oval electron-dense intranuclear droplets, exhibited arrays of small, electron-dense particles packed in the region of the sperm where one would expect to have encountered the spike, suggesting that these particles play a role in sperm spike depolymerization. It is suggested that the melanized condition reduces fecundity in this species by promoting the depolymerization of sperm spikes and the degeneration of sperm. The mechanism by which sperm and spermatophores become melanized is unknown and remains to be determined. No bacteria and no hemocytes were encountered in either light or electron microscope preparations of melanized spermatophores.

Ultrastructural evidence for the destruction of Schistosoma mansoni sporocysts associated with elevated lysosomal enzyme levels in Biomphalaria glabrata

The activity levels of serum acid phosphtase, aminopeptidase, and lysozyme in a Brazilian strain of Biomphalaria glabrata were ascertained at 1, 2, and 3 hr after mechanical wounding or injection with albumin on the 30th day postexposure to a compatible strain of Schistosoma mansoni miracidia and found to be elevated. Parallel transmission electron microscope studies on daughter sporocysts and developing cercariae at these time intervals revealed progressive disintegration of the parasites that was associated with increased numbers of host granulocytes abutting the sporocyst surfaces. Furthermore, host granulocytes were observed to have passed through eroded sporocyst walls and attacked developing cercarial embryos. It is proposed that the elevated levels of lysosomal hydrolases released from activated host granulocytes as a result of challenge altered the parasites surfaces so that these were recognized as nonself. Consequently, additional host granulocytic response, which included additional release of lysosomal enzymes into serum as well as phagocytosis of remnants of both sporocysts and developing cercariae, was elicited.

Light and electron microscope studies of smooth endoplasmic reticulum in dividing rot hepatic cells

Light and electron microscope studies of dividing hepatic cells of regenerating rat livers were performed in order to determine the location and intracellular associations of smooth endoplasmic reticulum during mitosis and cytokinesis. In contrast to an apparent random distribution during interphase, a definite association between centrosomal regions and smooth endoplasmic reticulum was observed in prometaphase. At metaphase and early anaphase, smooth endoplasmic reticulum was located between the poles of the mitotic spindle and the polar cell cortex. During late anaphase, smooth endoplasmic reticulum occurred in discrete subcortical arrays on either side of the spindle, and no longer at the poles of the cell. During early telophase, membranes of smooth endoplasmic reticulum were located in advance of the incipient cleavage furrow, while during late telophase, such membranes remained near the lateral cortex not involved in furrowing. Glycogen particles were admixed with smooth endoplasmic membranes during this sequence of events. Periodic acid-Schiff staining of thick sections revealed that masses of glycogen followed a pattern of distribution during mitosis and cytokinesis similar to that described above for smooth endoplasmic reticulum. These observations demonstrate distinct orientations and associations, and suggest displacement or flow, of smooth endoplasmic reticulum in hepatic cells during division. The possible role of smooth endoplasmic reticulum in regulating cytokinesis is discussed.

Junctional relationships between germinal cells and sustentacular cells in the testes of a palaemonid shrimp

Testes of the palaemonid shrimp Macrobrachium rosenbergii were prepared for study in the light, scanning and transmission electron microscopes and shown to be composed of solid, convoluted cords of tissue composed of two major sets of cells, spermatogenic and sustentacular cells. Among the spermatogenic cells, preleptotene spermatocytes and encysted spermatozoa were of most frequent occurrence. The sustentacular cells sent long, cytoplasmic extensions ramifying between and around tightly packed spermatocytes of the seminiferous cords and separated the spermatocytes from the basal lamina which surrounded the cords. Spermatocytes formed desmosome-like and short gap junctions with one another, while sustentacular cells formed intermediate-like junctions and gap junctions with spermatocytes. No special junctions between one sustentacular cell and another were encountered in the present study.

Ca-enriched amorphous mineral deposits associated with the plasma membranes of chondrocytes and matrix vesicles of rat epiphyseal cartilage.

SummaryElectron microscopic study of tibial epiphyseal plates of young growing rats revealed amorphous-appearing electron dense deposits 5–35 nm in diameter, associated with the plasma membranes of more than 43% of the proliferative zone chondrocytes. Hypertrophic zone chondrocytes, however, revealed no plasma membrane-associated amorphous-appearing deposits. The membrane-associated densities were observable in unstained sections of tissues fixed in glutaraldehyde alone and in tissues double-fixed with glutaraldehyde and osmium tetroxide, and were extracted from ultrathin sections floated on neutral aqueous solutions of 4% ethyleneglycol bis-(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA) for one-half hour. Energy dispersive X-ray analysis of the densities in scanning transmission electron microscope (STEM) mode revealed the presence of Ca, suggesting that the membrane-associated amorphous-appearing deposits are Ca-enriched. Similar deposits were observed in the membrane of matrix vesicles present in the longitudinal cartilaginous septae in the hypertrophic zone. Four types of matrix vesicles were encountered in the longitudinal cartilaginous septae; one type with amorphous-appearing deposits, another with crystallites, a third type with both amorphous-appearing and crystalline-like deposits, and a fourth that is empty. These observations are interpreted to indicate that chondrocytes of the reserve and proliferative zones play a direct role in mineralization by elaborating amorphous mineral deposits along their plasma membranes. These deposits are incorporated into budding matrix vesicles, which then play a role in the initiation of mineralization by supporting the spontaneous phase transformation of amorphous-appearing mineral to crystalline mineral.

Ultrastructural and histochemical observations on electroejaculated spermatophores of the palaemonid shrimp, Macrobrachium rosenbergii.

Spermatophores obtained by electroejaculation from mature males of Macrobrachium rosenbergii were studied by light microscopy and by transmission and scanning electron microscopy. Upon electrical stimulation, single spermatophores were usually extruded simultaneously from each gonopore and frequently became firmly adherent to one another forming a compound spermatophore. Individual spermatophores were pod-shaped. 0.5-1 cm long, 2-3 microm in diameter and consisted of a lateral sperm mass, a medial mucus mass, and a noncellular capsule. The capsule was a single-layered PAS-reactive structure composed of fine, intertwining fibrils and almost completely surrounded the sperm and mucus masses. On its outer surface were large. AB-reactive globules of flocculent material partially surrounded by finger-like extensions of a canaliculated reticulum composed of fine, intertwining fibrils. The canaliculated reticulum may permit fluid imbibition by the spermatophore. Forming a central band within the capsule were many small membrane-less AB/PAS-reactive granules. The lateral sperm masses consisted of unistellate spermatozoa embedded in an AB/PAS-reaetive matrix composed of globules of flocculent. AB-reactive material and a highly plastic canaliculated and thread-filled reticulum. The reticulum itself consisted of fine, intertwining fibrils. The threads within the interstices of the reticulum were long, curving structures, 30-36 nm in diameter. The function(s) of the canaliculated and thread-filled reticulum within the sperm mass is not known at this time. The medial mucus mass was similar in fine structure and histochemical reactivity to the sperm mass, except that spermatozoa were lacking. It is apparent that the spermatophore of M. rosenbergii consists of relatively few complexly arranged structural and macromolecular components designed to transport and protect the spermatozoa.

Ultrastructural changes in the secretory cycle of parathyroid cells of winter frogs (Rana pipiens) after pituitary homoimplantation

SummaryParathyroid glands of winter frogs (Rana pipiens) were compared by light and electron microscopy with those of winter frogs homoimplanted with pituitary glands. Serum calcium levels of untreated and pituitary-implanted animals were compared also. Forty-eight hours after pituitary implantation, serum calcium is elevated from a mean winter value of 6.2 mg % to 9.3 mg % and, morphologically, the parathyroid gland appears to be stimulated with respect to secretory activity. Compared with parathyroids of untreated winter frogs, intercellular spaces are diminished after pituitary implantation and glandular parenchyma is composed of cells with closely apposed plasma membranes thrown into interdigitating folds. Dense core vesicles are present in the cytoplasm and, together with microtubules, are encountered near plasma membranes. Golgi lamellae contain electron dense material and exhibit budding of dense core vesicles. Neither myelinated multivesicular bodies, presumably cytolysosomes degrading unneeded parathormone and organelles, nor focal dilatations with myelination of Golgi lamellae are encountered in parathyroid cells of pituitary implanted frogs. Rough endoplasmic reticulum and mitochondria do not undergo marked changes in distribution or abundance after pituitary implantation, indicating that the synthetic aspects of the secretory process are little altered in untreated and treated animals. It is suggested that in addition to Ca++ a pituitary factor is involved in the seasonal changes in amphibian parathyroid structure and function.

Zinc metalloprotease activity in the cement precursor secretion of the barnacle, Chthamalus fragilis Darwin.

Adult barnacles, Chathamalus fragilis, were removed carefully from the leaves and stems of marsh grass and floated base-up in algae-supplemented sea water. During the next 24-72 h, the animals secreted onto their exposed bases, a fluid, the cement precursor secretion (CPS), aliquots of which were collected in glass micropipets and pooled. The concentration of protein in pooled samples of CPS averaged 1.5 g +/- 0.42 protein/l of secretion. Protease activity was expressed as A(492) units/h/g of CPS protein. Aliquots of 5-45 l of pooled CPS samples, incubated in the presence of FTC-casein at 37 degrees C for 24 h, exhibited 8.31 x 10(-5) +/- 1.55 x 10(-5) DeltaA(492) units/hr/g of protein on average. Protease activity was optimized by the addition of 10 mM Ca(++) ions. Activity was detectable over a broad pH range, but was optimal around pH 8. Protease activity was inhibited up to 40% in the presence of 2.7 mM ethylenediaminetetraacetic acid (EDTA) and up to 97% in the presence of 2.5 mM 1,10-phenanthroline (OP) in the presence of 10 mM Ca(++) ions. Although low concentrations of Zn(++) ions (10 M) had little effect on protease activity, higher concentrations of Zn(++) ions (50 M to 15 mM Zn(++)) inhibited CPS protease activity. Protease activity was not inhibited by 1 mM phenylmethylsulfonylfluoride (PMSF), nor by 28 M E64, nor by 20 M leupeptin. At the present time, the protease activity in the barnacle CPS may best be characterized as a Ca-stimulated Zn-metalloprotease.