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The midgut epithelium of larval and early postlarval brown shrimp has been studied with light and electron microscopy. Ultrastructurally the features of the midgut do not change during these stages of development. On the basis of electron density, two epithelial cell types can be distinguished, and these are referred to as light and dark cells. The dark cells contain more rough endoplasmic reticulum and more free ribosomes than the light cells. Mitochondria in the dark cells have a matrix which is less electron dense than the mitochondrial matrix of the light cells. Both cell types have a microvillous border with a surface coat. The microvilli lack microfilaments within their core, and a terminal web is not differentiated in the stages examined. Tubular smooth endoplasmic reticulum is abundant in the basal portions of the cells. Electron dense, membrane bound vesicles are consistently seen in association with the Golgi apparatus, apical cell surface, and gut lumen and therefore are believed to be secretory granules. Cells in the anterior portion of the midgut often contain very large lipid droplets in the cytoplasm.

Fertilized mouse eggs regulate their size principally by accumulating glycine as an intracellular osmolyte using the GLYT1 (SLC6A9) transporter, a mechanism of cell volume homeostasis apparently unique to early embryos before the morula stage. However, nothing was known of cell volume regulation in oocytes before fertilization. We show here that GLYT1 is quiescent in mouse germinal-vesicle-stage oocytes but becomes fully activated within hours after ovulation is triggered. This initiates accumulation of substantial amounts of intracellular glycine in oocytes during meiotic progression, reaching a maximal level in mature eggs. Measurements of endogenous free glycine showed that there were nearly undetectable levels in ovarian germinal-vesicle-stage oocytes, but high levels were present in mature ovulated eggs and in preimplantation embryos through the two-cell stage, but not in morulae. Furthermore, intracellular glycine was regulated in response to changes in external tonicity in eggs and embryos through the two-cell stage, but not in oocytes or embryos after the two-cell stage. Before activation of GLYT1,oocytes were unable to independently regulate their volume. As GLYT1 became active, however, oocyte volume decreased substantially and oocytes gained the ability to regulate their size, which required GLYT1 activity. Before ovulation, oocyte size was instead determined by a strong adhesion to the rigid extracellular matrix of the oocyte, the zona pellucida, which was released coincident with GLYT1 activation. The ability to acutely regulate cell size is thus acquired by the oocyte only after ovulation, when it first develops glycine-dependent cell volume regulation.

Lobster aquaculture requires control of reproductive processes. We have previously described an electrical stimulation technique for collecting spermatophores from living lobsters (Homarus americanus). These spermatophores can potentially be used in a variety of ways including: 1) experimental manipulation of sperm, 2) artificial insemination of females, 3) sperm banking, and 4) evaluation of sperm production by the male. In this study, we evaluated spermatophore production and extrusion from: 1) laboratory reared H. americanus and H. gammarus, 2) stressed, wild-caught H. americanus, and 3) laboratory bred and reared hybrid males from reciprocal interspecific crosses. Our results show that the extrusion technique can be used successfully on H. americanus, H. gammarus and hybrid males. Laboratory reared H. americanus and H. gammarus from both Aquaculture Enterprises and Bodega Marine Laboratory extruded spermatophores in 60–85% of all trials. The majority of these spermatophores were of high quality containing large sperm masses with morphologically normal sperm. Stressed H. americanus, obtained from a market in Sunset Beach, California, 24 hours after shipment from New England, extruded fewer spermatophores which were shorter in length and inferior in quality. Hybrid males extruded spermatophores possessing a morphologically normal wall, but lacking sperm. These results demonstrate that this electrical stimulation technique can be used to screen males for spermatophore production and identify certain types of infertility.

Fertilization in mammals requires the successful completion of many steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion. In this minireview, we focus on three adhesion steps in this multistep process. The first is oocyte "pick-up," in which the degree of adhesion between the extracellular matrix of the cumulus cells and oviductal epithelial cells controls the successful pick-up of the oocyte-cumulus complex and its subsequent transfer into the oviduct. The second part of this review is concerned with the interaction between the sperm and the zona pellucida of the egg. Evidence is discussed that a plasma membrane form of galactosyltransferase on the surface of mouse sperm binds to ZP3 in the zona pellucida and initiates an acrosome reaction. Additional evidence raises the possibility that initial sperm binding to the zona pellucida is independent of ZP3. Last, we address the relationship between sperm adhesion to the egg plasma membrane and membrane fusion, especially the role of ADAM family proteins on the sperm surface and egg integrins.

Adhesion between the oocyte-cumulus complex and infundibulum plays an important, but poorly understood, role in oocyte pick-up. The purposes of this study were to determine which components of the oocyte-cumulus complex and oviductal epithelium function in adhesion, to measure adhesion under physiological conditions, and to examine the effect of modulation of adhesion on oocyte-cumulus complex pick-up rate. Oocyte-cumulus complexes containing an expanded matrix were readily transported into the oviduct, while unexpanded complexes lacking an extracellular matrix were not picked up, indicating that the matrix is necessary for pick-up. Transmission electron microscopy revealed that during pick-up, adhesion occurred specifically between the ciliary crowns of the oviduct and the granules and filaments of the cumulus matrix. An assay was developed using vacuum from a low-flow peristaltic precision pump, modified for bi-directional flow, to measure the strength of adhesion between the oocyte-cumulus complex and the oviductal epithelium, and adhesion was measured during physiological conditions. The lectin wheat germ agglutinin and the polycation poly-L-lysine were then used to modulate adhesion, and the effects of increasing or decreasing adhesion on oocyte pick-up rate and ciliary beat frequency were examined. The data show that 1) the matrix of the oocyte-cumulus complex and the ciliary crowns of the oviduct function in adhesion during pick-up and that adhesion is necessary for pick-up, 2) adhesion can be assayed quantitatively and is very uniform among control infundibula, and 3) decreasing or increasing adhesion decreases oocyte pick-up rate and in some cases prevents pick-up without affecting ciliary beat frequency.

Secondhand smoke (SHS) is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS), the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS) in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR) assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF) and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer. © 2014 Sarker et al.

The distribution of microfilaments and microtubules was examined in pleopod tegumental glands of male and female lobsters (Homarus americanus). Glands were labeled with rhodamine-phalloidin or antibodies to tubulin, the antitubulin antibodies being demonstrated with secondary antibodies conjugated to fluorescein. The labeled glands were then examined using either a Zeiss epifluorescence microscope or a Bio-Rad confocal scanning microscope. Some glands were examined using transmission electron microscopy. Glands from males and females showed the same distribution of microfilaments and microtubules, which appeared most abundantly in the common locus and around the main duct of each rosette. F-actin was specifically found in the central lobe of the central cell, around the ductules of the common locus, surrounding the finger-like projection of secretory cells, and encircling the main duct draining the rosette. Microtubules were most abundant in the finger-like projections of the secretory cells, in the cytoplasm of the central cell and canal cell, and around the main duct of the canal cell. Contraction of the microfilaments may facilitate movement of secretory product from the rosette, while the microtubules may provide structural support for the attenuated finger-like projections and the main duct. Electron micrographs suggest there is some interaction between these two elements of the cytoskeleton.

Preovulatory lobster oocytes were mechanically removed from follicles and fertilized in vitro with sperm from the vas deferens of males or the seminal receptacle of females. Oocytes were fixed for ultrastructural analysis at various times after insemination. Stages in gamete interaction beginning with sperm binding to envelope 1A and extending through nuclear envelope breakdown of the fertilizing sperm were examined microscopically. Numerous sperm bind to envelope 1A of mature oocytes. Only properly oriented sperm undergoing normal acrosome reactions penetrate envelopes 1A and 1B (together these form the vitelline envelope). During the acrosome reaction, the inner acrosomal material comes in direct contact with envelope 1B and does not appear to release a lysin for penetration of envelopes 1A and 1B. The acrosomal membrane overlying the acrosomal filament appears to be the first membrane of the sperm to contact the oolemma and is probably the initial site of gamete membrane fusion. Following fusion, egg cytoplasm flows around the sperm nucleus forming a small fertilization cone. The acrosomal contents are left outside the oocyte in the pervitelline space. Once gamete membrane fusion has occurred, the nuclear envelope fragments, and by 120 min after insemination, it has completely disappeared around some sperm nuclei. As sperm are incorporated into the ooplasm, the microtubules associated with the membrane/microtubule complex of the nucleus depolymerize, and the membranes of this complex are less frequently observed. After gamete membrane fusion, the acrosomal filament is contiguous with oocyte cytoplasm, and it remains structurally unchanged by 120 min after insemination. It is probable that the oocyte releases some high density cortical granules within the first 120 min of insemination; however, there was no evidence for the release of low density vesicles, ring vesicles, and moderately dense vesicles during this time.

The feasibility of fertilizing preovulatory lobster oocytes was examined in vitro under various experimental conditions. Large (1.6 mm diameter) and small (0.6 mm diameter) oocytes were compared in fertilization trials. Small oocytes were surrounded by thin envelope thought to correspond to envelope 1A of mature oocytes. Large oocytes were surrounded by a fully formed vitelline envelope comprised of two distinct sublayers, 1A and 1B. Although sperm bound very effectively to the coat surrounding small oocytes, none penetrated the coat or fertilized the oocytes. Large diameter oocytes that were removed from follicles by dissection were fertilized by sperm from the proximal vas deferens of males and by sperm from the seminal receptacle of females. Fertilized oocytes showed a high degree of polyspermy. Higher numbers of sperm bound to, penetrated, and fertilized large diameter oocytes when inseminations were done in a saline solution (2.5LSH) than when done in artificial sea water (ASW). Sperm failed to bind to large diameter oocytes that were induced to ovulate in vitro by collagenase treatment. Contaminating enzymes may have destroyed the sperm receptor in envelope 1A of collagenase treated oocytes. Our in vitro fertilization method will allow the process of fertilization to be studied experimentally in lobsters, and it may be applicable to other decapods.

Epidemiological studies have repeatedly shown that reproductive processes in pregnant women are adversely affected by exposure to cigarette smoke. The potential reproductive targets of smoke during pregnancy include the ovaries, oviducts, uterus, placenta, umbilical cord, and embryo/fetus. In vitro methods for studying the effects of smoke and its individual components have been developed and applied to each of these reproductive targets. In vitro assays have been useful in determining the biological processes that are affected in the reproductive organs and in identifying the cellular and molecular targets of smoke in each organ. In vitro methods have also been used to study the mechanism of action of smoke constituents, such as nicotine, on specific processes in reproductive organs and to screen smoke solutions to identify the molecules that affect reproduction. In general, data collected in vitro have confirmed, extended, and helped explain what has been learned from epidemiological studies. This review summarizes some of the in vitro assays that have been used to study cigarette smoke's effect on the nonpregnant and pregnant female reproductive tract and spotlights examples of their applications.

Fertilization in mammals requires the successful completion of many steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion. In this minireview, we focus on three adhesion steps in this multistep process. The first is oocyte "pick-up," in which the degree of adhesion between the extracellular matrix of the cumulus cells and oviductal epithelial cells controls the successful pick-up of the oocyte-cumulus complex and its subsequent transfer into the oviduct. The second part of this review is concerned with the interaction between the sperm and the zona pellucida of the egg. Evidence is discussed that a plasma membrane form of galactosyltransferase on the surface of mouse sperm binds to ZP3 in the zona pellucida and initiates an acrosome reaction. Additional evidence raises the possibility that initial sperm binding to the zona pellucida is independent of ZP3. Last, we address the relationship between sperm adhesion to the egg plasma membrane and membrane fusion, especially the role of ADAM family proteins on the sperm surface and egg integrins.

We hypothesized that reactive carbonyls generated from smoke exposure cause increased arterial low-density lipoprotein (LDL) accumulation and endothelial layer permeability. In addition, we hypothesized that estrogen supplementation was protective against chronic environmental tobacco smoke (ETS) exposure to the artery wall. Quantitative fluorescence microscopy was used to determine artery injury after exposure. For our chronic studies, ovariectomized rats treated with subcutaneous placebo or 17β-estradiol pellets were exposed to ETS or filtered air for 6 wk. ETS exposure increased carotid artery LDL accumulation more than fourfold compared with filtered air exposure, an effect largely mediated by increased permeability. No protective effect of estradiol was observed. Acute ETS exposure of a buffer solution containing LDL resulted in a more than sixfold increase in the highly reactive carbonyl glyoxal. Perfusion of this solution through carotid arteries resulted in a 105% increase in permeability. Moreover, perfusion of glyoxal alone caused a 50% increase in carotid artery permeability. This endothelial damage and changes in lipid accumulation may serve as an initiating event in atheroma formation in individuals exposed to ETS.

The muscular apparatus of the ovarian follicle has been investigated more thoroughly in eutherian mammals than in any other group of animals [1]. The mammalian ovarian follicle was first reported to contain contractile cells, in particular smooth muscle, over 100 years ago [2, 3, 4]. Because light microscopy does not resolve all the structural features of smooth muscle cells, controversy existed until recently regarding the type of contractile cell in the mammalian follicle [5]. During the past 30 years, however, there have been numerous ultrastructural studies that establish the existence of myoid cells with smooth muscle characteristics in ovarian follicles and numerous physiological studies that demonstrate that these follicles are contractile [6, 7]. Possible functions of the contractile apparatus include aiding in rupture site formation during ovulation [8, 9], evacuation of the antrum and collapse of the follicle wall during ovulation [6,10], corpus luteum formation [10], and compaction of the follicle during atresia [11]. Since comprehensive coverage of the literature in this field is beyond the scope of this review, the more recent work will be emphasized. Other reviews related to this topic have been published [1, 6, 7, 10, 12, 13].

Smooth muscle cells in the base of hamster follicles contract minutes prior to ovulation; during this contraction the profile of the follicle changes from low to tall. Our purpose was to measure intrafollicular pressure, independent of an intact circulatory system to the ovary, at the time when follicular smooth muscles are known to be contracting. Hamster ovaries were removed 13 hours after hCG injection and placed in a culture medium which supports in vitro ovulation. Intrafollicular pressure was measured continuously with a WPI Model 900 Micropressure system, while the follicle was being observed and photographed using a dissecting microscope and Zeiss 35 mm camera. Initial intrafollicular pressure was 2.1 +/- 1.1 mmHg (n = 47) in the absence of a blood supply. This pressure decreased slowly (0.03 mmHg/min.) until the transition from a low to tall profile occurred at which time the pressure decreased at a rate of 0.09 mmHg/min. At ovulation, the pressure drops precipitously to 0.0 mmHg. The condition of the follicle, in particular the apex, was examined using scanning electron microscopy (SEM). At 13 hours after hCG, rupture sites had started forming in the apices of 94.3% of the unovulated follicles. In some instances, surface epithelial cells were stretched apart and gaps were present between cells. More often portions of the surface epithelium were missing and the underlying cell layers were exposed. Fluid and erythrocytes were observed passing through the developing rupture sites. Taken together these observations demonstrate that intrafollicular pressure in hamster preovulatory follicles decreases gradually at the time follicular smooth muscle cells contract. We suggest that contraction of follicular SMC is a slow, gradual process which causes formation and/or enlargement of holes in the rupture site. An increase in the size or number of these apertures would lead to increased leakiness and could account for the observed acceleration in the rate of pressure decrease during profile transition. Our observations are the first to correlate changes in intrafollicular pressure with the contraction of smooth muscle cells and condition of the apex.

We have examined the formation of the egg attachment stalk in the lobsters Homarus americanus and H. gammarus. The formation of the stalk is similar in both species. Ovulated oocytes are surrounded by a single coat, envelope 1, composed of layers 1A and 1B. After passage through the gonopore and exposure to sea water, envelope 1 swells and becomes sticky. A second coat, envelope 2, forms between the oocyte and envelope 1 during a complex cortical reaction initiated after fertilization. Eggs pass over the ventral surface of spawning females to the region of the pleopods, where they stick by means of layer 1A to each other and to the ovigerous setae. Layers 1A and 1B are soft and pliable at this time. During egg attachment, the pleopods beat vigorously and cause envelope 1 to stretch and form attachment stalks. Beating probably also causes the attachment stalks to twist and wrap around the ovigerous setae. After the egg mass is secured to the ovigerous setae, envelope 1 of both the attachment stalk and egg coat condenses to form a tough material capable of securing the egg mass to the pleopods for intervals up to 16 months. After larvae hatch, portions of the egg coats and the attachment stalks are retained on the ovigerous setae until the female undergoes her next molt.

Because the acrosome of human sperm is too small to be directly visualized by phase-contrast microscopy, acrosome reactions (that is loss of the acrosome) are generally not evaluated in studies of human sperm capacitation and fertilization. Nevertheless, it would be useful in such studies to have a technique for easily identifying and quantitating acrosome-reacted sperm. In this paper, we describe a method for labeling the human sperm acrosome with fluorescein-conjugated Ricinus communis agglutinin-60 (FITC-RCA); we show that in sperm without acrosomal caps, FITC-RCA labeling occurs either not at all or only in the equatorial segment of the acrosome. To determine if the absence of FITC-RCA labeling in the acrosomal cap region gives a reliable estimate of acrosome reactions, washed sperm or sperm incubated in a capacitating medium (BWW) were divided into two groups, which were then fixed for FITC-RCA labeling or transmission electron microscopy. Counts of acrosome reactions made by each method were similar, and we observed an increase in the percentage of reactions following incubation in BWW. We conclude that the FITC-TCA labeling technique is a reliable method for accurately scoring the percentage of acrosome-reacted human sperm.

Freshly extruded spermatophores from the lobster, Homarus americanus, were examined using light microscopy and scanning and transmission electron microscopy. The tubular spermatophore is trifoil in shape with two lobes tapered laterally from a third lobe situated ventrally. It is comprised of sperm surrounded by three acellular investments: (1) a primary spermatophore layer, (2) an intermediate layer, and (3) an outer bounding layer. The sperm are packed into a continuous tube contained largely within the ventral lobe and are embedded in a matrix of moderate electron density. The primary spermatophore layer is uniformly thick around the sperm mass and contains at its peripheral margins both ring structures and crystals in an amorphous matrix. The intermediate layer is thicker dorsally than ventrally. Dense granules dominate the ventral half of the intermediate layer while inclusions populate the dorsal half; both react positively to the periodic acid-Schiff (PAS) technique. The innermost portion of the outer bounding layer is composed of parallel fibrils; a flocculent material is present peripherally. This flocculent substance is presumed to impart stickiness to freshly extruded spermatophores. These observations provide a basis for the future understanding of the mechanisms involved in long-term storage of sperm in spermatophores.

The role of contractile cells in mammalian ovulation is uncertain. In this study, we examined the morphology and distribution of cells within the theca externa of hamster follicles at various times during ovulation. Cells with all the ultrastructural features of smooth muscle (SMC) were found only in the basal hemisphere of the follicle. In contrast, the theca externa in the top half of the follicle was composed of fibroblasts. We next examined living hamster follicles during in vitro ovulation for morphological evidence of follicular contraction. The following changes in follicle shape were observed: (1) The base of the follicle moved apically; (2) follicles, which were initially spherical, became taller and thinner; and (3) after rupture, the apical follicle wall collapsed onto the surface of the ovary. To analyze the cause of these changes, sections of fixed follicles were examined by light and electron microscopy. During the final minutes before rupture, a V-shaped constriction formed in the base of the follicle. This constriction continued to narrow and by the time of rupture, it obliterated the basal part of the antrum. We concluded that the apical movement of the base of the follicle seen in living ovaries corresponds to the formation of the basal constriction seen in fixed sections. To determine if follicular SMC were involved in formation of the constriction, the ultrastructure of SMC was examined before, during, and after the constriction formed. The morphology of the SMC changed from the characteristic of relaxed or stretched SMC to that of contracted SMC when the constriction began to form. No other type of cell in the follicle wall showed these changes in morphology. We conclude that the formation of this constriction, and possibly the increase in height of preovulatory follicles and the collapse of the follicle wall after rupture, are due to contraction of SMC in the theca externa in the basal hemisphere of the follicle. This is the first morphological demonstration that follicular SMC contract prior to rupture of the follicle. The significance of these observations in mammalian ovulation is discussed.

Hamster sperm extracts contain a polypeptide which comigrates with muscle action on polyacrylamide gel electrophoresis. On double diffusion precipitation plates, sperm extracts form a single precipitin band with an antibody to muscle actin (antiactin) and show a reaction of identity with muscle actin. Indirect immunofluorescent microscopy revealed that antiactin binds along the concave margin and equatorial segment in the acrosomal region, in the connecting piece of the neck, and in the principal piece of the tail. These results are evidence that hamster sperm contain actin. The possible significance of these observations in fertilization is considered.

The morphology and kinetics of the normal acrosome reaction were examined in vitro using hamster sperm incubated in detoxified sera. The reaction involved either swelling and elevation or crenulation and fragmentation of the acrosomal cap. Swelling and elevation occurred during both normal and degenerative reactions, as reported by others. Crenulation with subsequent fragmentation of the cap was observed during normal reactions. Early crenulation of the acrosome could be induced by cold shock (5 degrees C, 25 minutes), but this did not decrease the incubation time required (at 37 degrees C) for completion of the normal reaction. In appropriate sera, the occurrence of normal and degenerative acrosome reactions in motile sperm was significantly separated in time to study the reactions independently. The duration of the normal reaction, i.e., the time between the first morphological change in the acrosome (initiation)until the actual detachment of the cap (termination) was estimated to be 20 minutes. Saline dilution of these sera delayed initiation of the reaction and increased the duration of the reaction once it had started. Data from cold-shock and serum dilution experiments indicate that the mechanisms which govern the initiation and termination of the normal reaction are independently variable, and further suggest that initiation involves a change in membrane permeability and that termination includes membrane vesiculation.

Ionophore A23187, in the presence of extracellular Ca+2, induces a morphologically normal acrosome reaction in sperm of the sea urchin and precocious acrosome reaction and activation of guinea pig sperm. Increased membrane permeability to Ca+2 is responsible for initiating the acrosome reaction in both sea urchin and guinea pig sperm. In sea urchin sperm, the permeability change is brought about by egg jelly, whereas in the guinea pig sperm it accompanies capacitation.

The spermatophore of the crayfish Pacifastacus leniusculus consists of two main parts: a sperm mass composed of sperm embedded in a dense fibrillar matrix and an acellular wall which surrounds the sperm mass and is formed from secretions produced in the vas deferens. Following extrusion from the male, the spermatophore wall, which is initially soft and sticky, undergoes a hardening process. In this study, the structure of the spermatophore walls of unextruded (soft) and hardened spermatophores were compared using light and electron microscopy. The wall of the unextruded spermatophore is composed of three concentric layers: a thin primary spermatophore layer which directly surrounds the sperm mass; a thick middle layer composed primarily of electron-dense, spherical granules; and a thick outer layer formed from a dense globular secretion. The primary spermatophore layer and outer globular layer are positive for carbohydrate with the periodic acid – Schiff method. Following extrusion and hardening, the walls of spermatophores showed several structural changes. These are (i) division of the middle granular layer into a compact inner region and a highly reticulated outer region; (ii) the loss of the outer globular layer; and (iii) the formation of a thickened ridge along one side of the spermatophore wall. The thickened ridge is fibrillar in structure and is believed to be derived from a structural modification of the outer globular layer. No structural modifications in the primary spermatophore layer were observed. We interpret these observations to indicate that the outer globular layer functions in attachment of the spermatophore to the female and the middle layer is involved in spermatophore hardening and sperm protection during storage.

We have developed a procedure for the long-term storage of lobster (Homarus) sperm. Sperm are collected from males by electrically stimulating the area around the gonopore. They are transferred with a bamboo stick to a plastic test tube containing paraffin oil and are stored for variable periods of time at 4–7°C. Sperm stored up to 289 days appear morphologically normal (equivalent to unstored sperm) when examined by phase contrast microscopy and electron microscopy, and morphologically normal sperm are able to undergo acrosome reactions. After longer periods of storage, degenerative changes begin to develop in sperm. These include loss of the nuclear spikes, condensation of the subacrosomal material, and distortion of the acrosome. Sperm stored better in spermatophores that had thick walls than in those with thin walls. In some spermatophores, bacteria were found in the sperm mass after storage; in general, sperm in these spermatophores were morphologically abnormal. This technique provides a means for saving lobster sperm for subsequent use in experiments or for artificial insemination of female lobsters. It may be adaptable to other invertebrate species that produce spermatophores.

A technique has been developed for artificially inseminating freshly molted female lobsters (Homarus). Using the criterion that eggs were fertilized if they showed development of normal cleavage patterns, at least 49.5% of the inseminated females extruded fertilized eggs. Sixty-two percent of the fertilized eggs developed to the eyespot stage, and 26.6% of these subsequently hatched. Eggs were not fertilized in 7.7% of the extrusions. In 42% of the samples, it was not possible to establish whether fertilization had occurred because either samples were collected prior to initiation of cleavage or eggs were too poorly preserved to assess cleavage with confidence. It is probable that many of the eggs that were evaluated as equivocal were also fertilized. This technique should be valuable in the experimental and commercial aquaculture of lobsters and might be directly applicable to other crustaceans.

The fine structure of mature sperm from the spiny lobster (Panulirus) has been examined. Each sperm comprises a nucleus, lamellar region, and acrosome. The nucleus (Feulgen-positive region) contains uncondensed chromatin and is limited by a nuclear envelope. A variable number of spikes extend from the nucleus. The spikes are lined by evaginated nuclear envelope and are packed with microtubules, which span the nucleus proper. The spikes are stationary, and the sperm are immotile. The lamellar body, which lies at one side of the base of the acrosome and external to the nuclear envelope, contains numerous stacks of membrane and small mitochondria-like bodies. The acrosomal vesicle (PAS-positive region) is structurally complex and can be divided into four discrete zones, which are homogeneous, scrolled, crystalline, and flocculent in appearance. The vesicle is surrounded by periacrosomal material which is flocculent near the base of the acrosome and filamentous at the apex. The structural features of Panulirus sperm are compared to those of other reptantians (lobsters, crayfish, crabs) and natantians (prawns, shrimp), and it is concluded that at least two basic structural plans for decapod sperm exist.

The apical regions of maturing hamster follicles were examined by electron microscopy at 14, 9, 5, and 3 h prior to expected ovulation. At all time periods, a discrete layer of smooth muscle cells was found within the theca externa of the maturing follicle. In addition, bundles and clusters of filaments were found in the surface epithelial cells of maturing hamster follicles throughout the preovulatory period. A contractile role in ovulation is suggested for both the smooth muscle and surface epithelial cells.

The effect of several hydrolytic enzymes on ovulation of lobster (Homarus americanus) oocytes was studied in vitro using strips of ovarian tissue. Only ovaries which were judged by candling and microscopic examination to be close to natural ovulation were used. Ovaries were removed and cut into small, flat strips (1 cm2) and placed in either seawater (SW) or SW containing an enzyme. The number of oocytes ovulated from these strips was determined periodically over a 3–6-hour period. Of the enzymes tested, only crude collagenase treatment produced ovulation. Highly purified collagenase was tested subsequently and found effective. Moreover, when cysteine, a collagenase inhibitor, was added to crude collagenase preparations, ovulation did not occur. These results (1) show that mature lobster ovaries will undergo in vitro ovulation in SW solutions containing collagenase, and (2) are the first demonstration that a hydrolytic enzyme may be directly involved in invertebrate ovulation. It is suggested that collagenase disrupts the thick basement membrane surrounding each follicle, thus initiating follicular rupture and subsequent extrusion of the oocyte. This technique provides a useful method for in vitro studies of lobster ovulation.

Changes in the morphology of epithelial cells covering the sides and apex of ovarian follicles were examined in mice and hamsters during the final 13 hr before rupture using light and electron microscopy. At the time of the surge of luteinizing hormone, approximately 13 hr before follicle rupture, epithelial cells along the follicle sides are spherical, covered with microvilli, and remain so throughout the entire cycle. As ovulation approaches, cells at the apex become progressively flatter, increase in diameter, and undergo a reduction in the number and length of microvilli. By 2 hr before ovulation, the microvilli are present only along the boundary between adjacent cells and the cells are in different stages of degeneration. In some cells, the cytoplasm is electron dense and the nuclei are pyknotic. Other cells become electron lucent and cytoplasmic elements are leached from the cell. The apical plasma membrane is lost first over the center of the cell and later over the periphery. Epithelial cells detach from the apex individually until a large patch devoid of cells is formed. This includes the site of eventual rupture. The loss of epithelial cells from the apex of ovarian follicles of other species is compared with our results, and the processes involved in stretching, degeneration, and sloughing of the epithelial cells are discussed.

An experiment was conducted to examine the condition of the penaeid reproductive tract at various times after introduction into indoor tanks. Penaeus setiferus were collected off the coast of Corpus Christi during June and placed in 3.7-m diameter fiberglass holding tanks. After 1, 35, 49, 53, 76, and 131 days, the reproductive tracts were removed and compared using a combination of microscopical techniques. By day 35, the ampoules (segment 4 of the vas deferens) lacked turgidity, were slightly brown and contained little secretory product. Experimental spermatophores exhibited darkening and progressive loss of the wing. The ventral wall of the sperm sac was partially lost by day 35 and missing by day 49. The sperm mass of experimental spermatophores contained dead sperm by day 35 and numerous bacteria by day 49. Only the glutinous mass of the spermatophore appeared normal, although sometimes reduced in size, at all experimental times. In most day-35 and day-49 males, segments 1–3 of the vas deferens appeared normal with the dissecting microscope. However, when examined with phase contrast microscopy or scanning electron microscopy, sperm from segment 2B were dead, and the sperm mass contained bacteria. By 76 and 131 days, all tracts showed yellow discoloration of the sperm mass in segment 2. The testes in day-1 males were white and turgid; at all other times they were either partially or completely translucent. All males were infertile by day 35, our earliest experimental time, even though the vas deferens usually appeared normal at the level of the dissecting microscope.

The role of intrafollicular pressure in evacuation of the hamster cumulus from the follicle at ovulation was investigated experimentally in vitro. Previous work has shown that the cumulus is generally evacuated in two stages (Talbot, '82). Phase 1 occurs immediately at rupture and is characterized by rapid (

The numbers of eggs spawned, attached, and carried through to hatching were examined in three groups of captive lobsters ( Homarus americanus ). Wild born/lab spawned H. americanus , lab born/lab spawned H. americanus , and hybrids of H. americanus ♂ × H. gammarus ♀ spawned 8800–11500 eggs per female (weight range 336–945 g). Hybrid females attached 77% of all spawned eggs. Purebred females attached either most eggs of a spawn or fewer than 10%. In some instances where attachment was poor, females were known to be virgins or had been mated to a sterile hybrid male. Unfertilized eggs could attach, but the percentage which did was low. Some wild born/lab spawned H. americanus carried portions of their clutches through until hatching; however, many purebreds and all hybrids dropped their clutches before hatching. Possible factors which cause eggs to drop prematurely are discussed.

The wall of hamster ovarian follicles is composed of the following cell layers: granulosa, theca interna, theca externa, and the surface epithelium. To determine the morphological changes occurring in the follicle during formation of the rupture site, we measured: the thickness of each layer, the number of cells in each layer, and size of cells in each layer, at the apex and base of follicles at specific times during the final 13 hr before ovulation. Changes in the wall occur in 3 stages. During stage 1 (T0-T8), which includes the first 8 hr following the surge in luteinizing hormone, the apical and basal walls thin at the same rate and the antrum increases in size. During stage 2 (T9-T12), there is no change in the thickness of the apical and basal follicle wall nor in the size of the antrum. During the third stage (T12-T13), the size of the antrum decreases slightly and there is an increase in the thickness of the basal wall, which is correlated with its constriction caused by the contraction of smooth muscle cells. The apical wall rapidly thins to the point of rupture. The morphology of cells from each layer is described. Theca interna cells form the final tissue preventing escape of the oocyte-cumulus complex. The roles of cell migration, stretching, and death in thinning of the apical wall are described, and the mechanisms involved in follicle rupture are discussed.

The structure of the hamster oocyte-cumulus complex (OCC) has been analyzed to help understand how mammalian sperm penetrate the investing coats of the oocyte. At ovulation, oocytes of most mammalian species are surrounded by a zona pellucida, corona radiata, and cumulus layer. Cells of the cumulus and corona radiata are separated by an extracellular matrix (ECM) that contains hyaluronic acid. In hamsters, the diameter of mature follicular OCCs is 0.61 ± 0.12 mm, whereas in freshly ovulated OCCs in culture medium it is 0.78 ± 0.15 mm. This indicates the OCC expands at or after ovulation. The corona radiata is 1–4 cell layers deep, and corona radiata cells are closely packed (center-to-center distances between adjacent cells in follicular OCCs averaged 14 ± 3 μm). The cumulus layer is 5–8 cell layers deep, and intercellular spaces are much larger (center-to-center distances between adjacent cumulus cells in follicular OCCs averaged 50 ± 20 μm). An ovulated OCC has an approximate volume of 248 × 106 μm3 and was estimated to contain approximately 5,700 cells. Studies with stretched OCCs show that ink particles can readily penetrate the extracellular spaces of the cumulus and corona radiata layers and interact with the ECM, staining it black. At the periphery of the OCC, the ECM appeared discontinuous and formed “cords” containing cumulus cells, whereas closer to the corona radiata the matrix completely filled intercellular spaces. Ink penetrated the corona radiata, but the ECM was difficult to visualize because of the close packing of cells in this layer. Our observations on unfixed OCCs show that cell packing becomes more dense proceeding from the periphery of the OCC to the zona pellucida and that the ECM at the periphery differs from the matrix deeper in the OCC. These data suggest that an incoming sperm would find penetration of the investing coats to increase in difficulty (that is physical resistance would increase) as it got closer to the oocyte. We have also examined the effects of processing for microscopy on the structure of the OCC. Both standard fixation procedures and fixation in the presence of ruthenium red caused condensation of the ECM, especially in the cumulus layer, and thus produced a significant decrease in the diameter of the OCC.

A procedure is described for quantifying the percentage of normal acrosome reactions in fixed mouse sperm. This technique, which is a modification of the triple stain technique (TST) devised for human sperm [Talbot and Chacon, 1981a, b], utilizes trypan blue exclusion to identify the percentage of dead sperm in culture and subsequent staining of fixed sperm to differentiate the acrosome. Four classes of mouse sperm can be distinguished with the TST: (1) “live,” unreacted sperm, (2) “live,” acrosome-reacted sperm (normal reactions), (3) dead, unreacted sperm, and (4) dead, acrosome-reacted sperm (degenerative acrosome reactions). We have used this technique to study the time course of the normal acrosome reaction of mouse sperm in vitro. Our results with TST show that a low percentage of mouse sperm undergo normal acrosome reactions during the first 30 min of incubation; a maximum level of reactions is reached by 2 hr. Calcium was required in the medium for occurrence of normal reactions.

We have analyzed the structure of the mature ovarian follicle from the lobster, Homarus americanus. Mature follicles are composed of five parts which from inside to outside are: (i) the oocyte, (ii) the chorion, (iii) the follicle cells, (iv) a thick basal lamina, and (v) cells associated with the follicle's surface. The oocyte comprises most of the mass of the follicle; it is packed with yolk and green in color due to the presence of ovoverdin. The oolemma forms thick microvilli which project into the perivitelline space and through the chorion or oocyte investing coat. The chorion is composed of two regions, an endochorion containing “bottlebrush” inclusions and a thinner exochorion. The chorion is completely surrounded by a single layer of follicle cells which are polygonal in shape. The follicle cells exhibit at least four types of synthetic activity. The components of the endochorion are synthesized in smooth vesicles in the supranuclear region of follicle cells. Rough endoplasmic reticulum, Golgi bodies, and associated electron-dense granules are present throughout the cells; the granules appeared to be released at the cell's basal surface and contribute to chorion formation. An extensive network of tubular smooth endoplasmic reticulum and associated vesicles containing moderately electron-dense material was present in the subnuclear zone; the organization of this network suggests the follicle cells are active in steroid synthesis. Lamellar bodies composed of alternating electron-dense and lucid bands were common, especially in the basal cytoplasm; their function is not yet known. The entire follicle is surrounded by a thick basal lamina. Often this basal lamina forms the outermost surface of the follicle. However, in some regions it was covered by surface cells including fibroblasts, blood vessels, muscle cells, hemocytes, and epithelial cells. This description of the ultrastructure of the lobster ovarian follicle will provide a framework for future investigations on ovarian physiology.

Capacitated guinea pig sperm are more agglutinable by the lectin soybean agglutinin (SBA) than uncapacitated sperm (Talbot and Franklin, '78). This study demonstrates that uncapacitated guinea pig sperm become as agglutinable by SBA as capacitated sperm when treated with trypsin, but not chymotrypsin. The pattern of lectin induced sperm agglutination after trypsinization resembles that for capacitated sperm. Also, trypsinization specifically increases SBA induced agglutination and does not affect agglutination by RCA-60; similar results are obtained during in vitro capacitation. Taken together, these data may indicate that a trypsin-like enzyme modifies the sperm surface during capacitation.

Mammalian sperm must pass between cumulus cells and corona radiata cells before reaching the surface of the zona pellucida which surrounds the oocyte. The cumulus and corona radiata cells are separated from each other by an extracellular matrix (ECM) containing hyaluronic acid. The structure of this ECM and of the zona pellucida was investigated in the hamster oocyte-cumulus complex (OCC) using transmission electron microscopy (TEM) following processing in ruthenium red. When fixed in the presence of ruthenium red, the ECM of the OCC and the zona pellucida were well preserved and highly structured. The ECM between corona radiata cells was comprised of a network of granules and filaments which resembled hyaluronic acid containing matrices described in other systems. The outer one-third to one-half of the zona pellucida was porous; the ECM of the corona radiata extended into these pores. Bovine testicular hyaluronidase, Streptomyces hyaluronidase, and hamster sperm extracts containing hyaluronidase each dispersed the cumulus cells and most of the corona radiata cells. TEM examination revealed that brief (5-10 min) hyaluronidase treatment of OCCs removed the matrix filaments and caused clumping of the granules in both the corona radiata and zona pellucida. Longer hyaluronidase treatments (15-30 min) removed both filaments and granules. Our observations are consistent with the ideas that: 1) the ECM between corona radiata cells contains hyaluronic acid, and 2) hyaluronic acid is present in the outer one-third to one-half of the zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the anatomy of the vas deferens of the lobster Homarus americanus and have described the structure of the proximal vas deferens (segments one and two). The two tubes of segment one descend from the testes and gradually merge into segment two. The epithelium of segment one has synthetic activity and appears to contribute to the sperm-supporting matrix by exocytotic release of granules through its apical surface. The epithelium of segment two is also highly synthetic and secretes the primary spermatophore layer and part of the intermediate layer that surround the sperm mass. The trifoil shape of the extruded spermatophore is established through a change in height of some of the cells lining the lumen in segment two. Connective tissue and circular bands of striated muscle surround the epithelium of both segments.

Epibiotic bacteria on eggs from 66 female lobsters ( Homarus ) were examined to determine if these flora were causally related to loss of eggs from the pleopods. Eggs from five categories of berried female were examined. These included (1) wild born/wild spawned H. americanus , N =11, (2) wild born/lab spawned H. gammarus , N =16, (3) wild born/lab spawned H. americanus , N =18, (4) lab born/lab spawned H. americanus , N =10, and (5) laboratory bred hybrids of H. gammarus × H. americanus crosses, N =11. Four morphological types of bacteria were observed. These are: (1) filamentous bacteria, primarily Leucothrix -like, (2) disc-shaped bacteria resembling Microcyclus , (3) colonial rods, and (4) other rods which attached to surfaces by one end only. Wild born/wild spawned H. americanus eggs had all four types of epibionts. Egg coats had a higher number of total bacteria than stalks, including numberous colonial rods, but fewer filaments and other rods. Samples from laboratory spawned H. americanus contained all four types of bacteria with no distinction noted between egg coat and stalks. H. gammarus eggs had fewer surface bacteria than all other samples; these eggs were consistently occupied by a large number of discs, but were without filaments. Hybrid egg samples resembled H. americanus in both the number (high) and diversity (all four) of epibiotic bacteria on egg coat and stalks. Observations showed that: (1) wild spawned eggs can develop a high bacterial load yet still develop normally and be retained, (2) laboratory spawned eggs may drop off for reasons independent of epibiotic growth, or may be retained even when epibiotic growth is severe, or may drop off as epibiotic bacteria establish themselves on coat/stalk surfaces, (3) H. gammarus eggs have relatively clean egg/stalk surfaces and were retained well, (4) hybrid females have eggs resembling those from lab spawned H. americanus females both in epibiotic composition and proclivity toward drop off.

Aflagellate sperm of the crayfishes Cherax tenuimanus and Cherax albidus are composed of an acrosome, subacrosomal region, collar, and nucleus. The acrosomes are helmet-shaped and PAS positive. In C. tenuimanus, they can be subdivided into a granular inner region and a homogeneous outer region. A series ofelectron-translucent canals runs through the acrosomes of both species. In C. albidus, these canals are arranged in whorling patterns, while in C. tenuimanus they are present near the base of the acrosome and contain a small electrondense component. The subacrosomal regions are metachromatic when stained with methylene blue and contain a network of fibrils and granules. The collars are composed of granular material, which is positioned immediately subjacent to the subacrosomal region, and of highly convoluted membrane lamellae. Mitochondria and microtubules are associated with the membrane lamellae. The nuclei contain uncondensed chromatin and are bounded by a complete nuclear envelope. Sperm are surrounded by a moderately electron-dense matrix containing granules and filaments. Sperm of Cherax tenuimanus have large deposits of an electron-dense material on their surface. In contrast, sperm of C. albidus have long extracellular lamellae associated with the extracellular surface of their plasma membrane, and these lamellae are also found in the extracellular matrix surrounding the sperm of C. albidus. In both species, the sperm and extracellular materials are surrounded by a moderately PAS-positive primary spermatophore layer which is in turn surrounded by a secondary spermatophore layer secreted from the wall of the vas deferens. This layer includes dense granules which are supported in a matrix. In C. tenuimanus, this matrix is homogeneous, while in C. albidus, this matrix contains light and dark staining areas. The darker regions often have a fibrous appearance. A comparison is made between sperm of C. tenuimanus and C. albidus and the sperm of other pleocyematan species. Cherax tenuimanus (marron) and Cherax albidus (koonac) are fresh-water crayfish native to the large streams of southwestern Australia. Cherax tenuimanus is the third largest species of crayfish in the world (the Tasmanian crayfish Astacopsis franklinii and the Murray River crayfish Euastacus serratus are larger), and attains weights of approximately 2 kg (Shireman, 1973). Cherax albidus is smaller than C. tenuimanus, yet is still much larger than the native American genera (Pacifastacus, Procambarus) which weigh less than one fiftieth of a kilogram. Cherax tenuimanus and C. albidus are excellent animals for aquaculturing, because, in addition to their large size, they are highly palatable, do not burrow, have an uncomplicated life cycle, and grow large fairly rapidly (Crook, 1981). Their potential for aquaculture has generated considerable recent interest in these species. A complete understanding of the reproductive biology of species of Cherax will aid in their aquaculture. Until now, the ultrastructure of their gametes has not been studied, though work has been done on the ultrastructure of sperm from other crayfish genera (Moses, 1956, 196 la, b; Meek and Moses, 1961; Yasuzumi et al., 1961; Yasuzumi and Lee, 1966; Anderson and Ellis, 1967; Pochon-Masson, 1968; Lopez-Camps et al., 1981; Dudenhausen and Talbot, 1982). However, in many of the investigations on American crayfish, morphological detail was not completely resolved because of fixation problems. In this study, we have used the improved fixation procedures of Dudenhausen and Talbot (1982) to examine and compare the ultrastructure of sperm from C. tenuimanus and C. albidus.