bluuebunny/crossref_metadata_2025 huggingface dataset:
Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs
Authors: Fraser Lynn R.
Published: February 2005

Extracellular Ca2+ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca2+. In this study, Sr2+, Ba2+, and Mg2+ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba2+ proved to be ineffective, but Mg2+‐containing medium was able to support capacitation to a greater extent than unsupplemented Ca2+‐deficient media; despite this, Ca2+ was required for fertilization. In contrast, Sr2+ proved capable of substituting for Ca2+ in all events. Furthermore, Sr2+‐induced responses were indistinguishable from the corresponding Ca2+‐induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr2+ required the use of zona‐free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr2+. When zona‐intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr2+ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr2+ can profoundly affect both gametes in the mouse: It substitutes completely for Ca2+ in sperm responses and rapidly activates eggs, possibly by displacing Ca2+ from intracellular stores into the cytoplasm, where the Ca2+ can then trigger the various events of activation.
A complex cortical reaction leads to formation of the fertilization envelope in the lobster, Homarus
Authors: Talbot P. , Goudeau Marie
Published: February 2005

We have examined the formation of the fertilization envelope in the lobsters Homarus americanus and H gammarus. Oocytes were fixed for electron microscopy either in the ovary or following extrusion from the gonopore. Mature ovarian oocytes are surrounded by a coat (envelope 1), which is comprised of small electron‐dense granules and structures resembling “bottlebrushes.” At least part of this coat is synthesized by the follicle cells of the ovary. The cortex of ovarian oocytes contains four types of vesicles that we refer to as high‐density vesicles (HDV), low‐density vesicles (LDV), moderately dense vesicles (MDV), and ring vesicles (RV). Oocytes that were electrically extruded from the gonopore and fixed immediately had an envelope identical to that of ovarian oocytes. The cortex of gonopore oocytes contained the four types of vesicles found in ovarian oocytes. When unfertilized gonopore oocytes were allowed to incubate in sea water, the oocyte cortex appeared unaltered, but envelope 1 swelled and the bottlebrushes dispersed. When recently fertilized oocytes were fixed during natural spawning or following in‐vitro fertilization, each type of vesicle was released in sequence from the cortex of the oocyte. The contents of the HDV and LDV appeared first in the perivitelline space, but their fate could not be determined at later times. The ring‐shaped elements of the RV and the moderately electron‐dense material of the MDV were released exocytotically somewhat later; these materials coalesced in the perivitelline space to form a new coat (envelope 2). Envelope 1 subsequently condensed to its original thickness and appeared firmly attached to envelope 2. Our results show that the fertilized lobster egg is surrounded by two discrete coats. The outer coat, which is formed in the ovary, undergoes a swelling/condensation cycle at spawning. The inner coat originates from a complex cortical reaction. Together these coats comprise the fertilization envelope of the lobster egg.
Rhesus monkey sperm penetration into zona‐free hamster ova: Comparison of preparation and culture conditions
Authors: Binor Zvi , Rawlins Richard G. , Van der Ven Hans , Paul W. Dmowski
Published: February 2005

A major problem in development of nonhuman primate in vitro fertilization is the selection of donor males and repeated collection of consistent sperm samples. In practice, collection of a viable semen sample is highly dependent on operator technique and the type of animal restraint. We report an updated method for semen collection from the rhesus monkey (Macaca mulatta), use of TES‐Tris (TEST) Yolk Buffer (TYB) for prolonged sperm storage and improved results of hamster ovum penetration assay. Semen was obtained from adult males restrained with 2.0 mg/kg IM ketamine hydrochloride prior to direct penile stimulation (Grass SD‐9, frequency 150, delay 9, duration 7, volts 12–18, repeat mode, twin pulse). Liquified semen was washed and centrifuged twice at 100 × g for 5 min in BWW, Ham's F‐10 and TALP and allowed to swim‐up 60 min at 37° in 5% CO2 and air. Alternatively, semen was mixed 1:1 with TYB, refrigerated 20 h at 4°C, centrifuged at 100 × g for 5 min, and the pellet resuspended in 1.0 ml of TALP or BWW prior to use. Hamster ova penetration was achieved with capacitated macaque sperm. Penetration was significantly improved (P < .001) with preincubation in TYB followed by resuspension in TALP (79%).
A glycoprotein of oviductal origin alters biochemical properties of the zona pellucida of hamster egg
Authors: Oikawa Taneaki , Sendai Yutaka , Kurata Sho‐Ichiro , Yanagimachi Ryuzo
Published: February 2005

Eggs were isolated from ovaries and oviducts of the golden hamster and the components of zonae pellucidae were examined using density gradient SDS‐polyacrylamide electrophoresis. Zonae of ovarian eggs (ZP‐OVA) had three major components corresponding to the so‐called ZP‐1, ZP‐2, and ZP‐3. Zonae of recently ovulated eggs collected from oviducts (ZP‐OVI) had a 200–240 K component (ZP‐O) in addition to the three components present in ZP‐OVA. When ovarian and oviductal eggs were stained with FITC‐conjugated B. simplicifolia‐1 lectin (BS‐1), which specifically binds to alpha‐D‐galactose‐ or alpha‐N‐acetyl‐D‐galactosamine‐like terminal saccharide residues, ZP‐OVI was intensely stained, while ZP‐OVA was not. ZP‐OVA gained the ability to bind to BS‐1 after a brief treatment with oviduct extracts. These results suggest that biochemical properties of hamster zonae change after transport of eggs from ovary to the oviduct. The addition of the 200–240 K component of oviductal origin to preexisting zona components seems to be responsible for this change.
Effects of delayed mating on preimplantation embryos in spontaneously ovulated mice
Authors: Sakai Nobuyoshi , Endo Akira
Published: February 2005

The effects of delayed mating on mouse preimplantation embryos (78 ± 1 hours) were studied by setting up different mating periods in relation to the estimated time of spontaneous ovulation. Copulation occurred even in the late morning and early afternoon after the night of spontaneous ovulation. However, females mated in the early afternoon had no viable embryos at the time of laparotomy. Although embryonic development was not affected in the groups mated 6 or 10 hours after estimated ovulation, the percentage of degenerated embryos was increased in these groups. These results suggest that prolonged intervals between the estimated time of ovulation and mating have some deleterious effects on preimplantation embryos.
Characterization of actin isoforms in ejaculated boar spermatozoa
Authors: Casale Antonella , Camatini Marina , Skalli Omar , Gabbiani Giulio
Published: February 2005

Actin was localized in boar ejaculated spermatozoa by using specific antisera against cytoplasmic isoforms of actin [Otey et al., J Cell Biol, 102:1726–1737, 1986; Skalli et al., J Cell Biol, 103:2787–2796, 1986; Miller et al., Biochemistry, 26:6064–6070, 1987]. Indirect immunofluorescence and immunoelectron microscopy showed that γ and β actins were codistributed in the acrosomal and postacrosomal regions. Sperm actin was also identified on two‐dimensional gel as two spots in the isoelectric point and molecular weight corresponding to β and γ actins. Coelectrophoresis of sperm actin and chicken gizzard actin and immunoblots stained with the specific antibodies confirmed the presence of these two isoforms of actin.
Apparent densities of spermatozoa of various mammalian species
Authors: Oshio Shigeru
Published: February 2005

The apparent densities of sperm cells in several mammalian species were determined by means of Percoll density‐gradient centrifugation. Semen samples were obtained from (1) ejaculates from the bull, human, and rabbit, and (2) from the cauda epididymis of the goat, golden hamster, house musk shrew, mastomys, and mouse. The profiles of sperm distribution showed a single peak after the centrifugation in the first group and goat, whereas two separate main peaks in the second group (except goat). This disparity in sperm distribution profiles may be due mainly to differences in the degree of maturity of the sperm. Highly motile and mature sperm were obtained at higher densities, whereas immotile or immature sperm were found at the apparent densities.

Thus, in mammals, the profiles of sperm distribution in the Percoll density gradient are classified into two types, those with a single peak and those with two separate peaks. The apparent density of sperm cells may be of importance in sperm physiology.
Classification, inhibition, and specificity studies of the vitelline coat lysin from toad sperm
Authors: Yamasaki Hisashi , Takamune Kazufumi , Katagiri Chiaki
Published: February 2005

The sonicated supernatant of the sperm of the toad, Bufo japonicus, can digest easily the vitelline coat (VC) of uterine eggs, and to a lesser extent the VC of coelomic eggs, but not that of activated eggs. The VC lysis and fertilization were competitively inhibited in the presence of t‐butyloxycarbonyl‐L‐Gln‐L‐Arg‐L‐Arg‐4‐methylcoumaryl‐7‐amide (Boc‐Gln‐Arg‐Arg‐MCA), suggesting the involvement of proteases in the fertilization process. Starting from a sonicated supernatant, a potent VC lysin, possessing hydrolytic activity on Boc‐Gln‐Arg‐Arg‐MCA, was obtained by anion‐exchange chromatography and gel filtration. The activity of the partially purified lysin was inhibited by diisopropyl fluorophosphate (DFP) and by such trypsin inhibitors as soybean trypsin inhibitor, leupeptin, and (p‐amidinophenyl) methanesulfonyl fluoride hydrochloride, but not by chymostatin, E‐64, and ethylene glycol bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid. The molecular weight of the lysin was estimated to be 32K, based on the fluorographic image of 3H‐DFP binding to the lysin on sodium dodecyl sulfate gel electrophoresis. The VC lysin was most active at pH 7.0–7.6 and under low ionic strength equivalent to fresh water. The release of the VC lysin was induced upon incubation of sperm with the contents of oviducal pars recta granules (PRG), which are known to induce the acrosome reaction. We conclude that the protease studied here represents the VC lysin of toad sperm that is involved in fertilization by digesting the VC of uterine eggs, probably released as a result of the acrosome reaction induced by PRG.
Cytology of lepidoptera. III. Giant cysts: A morphological trait of apyrene spermatogenesis in an Ephestia kuehniella strain
Authors: Wolf Klaus Werner , Baumgart Kirsten , Traut Walther
Published: February 2005

A comparative investigation of testicular eupyrene cysts (in larvae) and apyrene cysts (in pupae) of Ephestia kuehniella laboratory strains was conducted using light and electron microscopy. Eupyrene cysts in the first meiotic division contained 64 spermatocytes, which showed only moderate asynchrony. In one of the strains, a wild‐type strain, L, normal‐sized cysts occurred together with abnormally large cysts. These are called giant cysts in this article. One of the premeiotic cysts, early giant cysts, studied in detail, contained approximately a fourfold number of cells compared with the number in a eupyrene cyst of the same stage. In cysts harboring spermatocytes and spermatids, late giant cysts, cell differentiation was highly asynchronous.

Failure in one of two control mechanisms in early cyst development may have caused the appearance of the cysts. Control of cell division might have been sloppy in apyrene spermatogonia. Hence, the spermatogonia within the cyst could have passed through additional division cycles. Alternatively, the giant cysts may have originated from more than one predefinitive gonial cell enclosed in a common envelope of sheath cells. As a third possibility, giant cysts could have arisen by fusion of normal cysts at a later stage. In either case, this is evidence that separation of eupyrene and apyrene pathways is earlier than was previously expected.

In two other Ephestia strains, apyrene sperm development proceeded without formation of giant cysts. One was a mutant strain, a, and the other one was a recently established wild‐type strain, Sbr. Apyrene sperm development is considered an example of degenerate evolution in which enhanced variability between species and even between populations of one species is a common phenomenon.
Trout sertoli and leydig cells: Isolation, separation, and culture
Authors: Loir Maurice
Published: February 2005

Trout testes at various stages of maturation were dissociated by perfusion at 12°C with collagenase plus pronase and then with collagenase alone, followed by slight shaking overnight in 1% bovine albumin. This step provided a suspension of isolated somatic and germ cells, clusters of interstitial cells, and either intact spermatogenetic cysts (meiotic testes) or clusters of Sertoli cells (other testes). Most of the spermatozoa were removed from the testis cell suspension by centrifugation in Percoll (density 1.065 g/ml). Sertoli and Leydig cells were prepared by a two‐step separation method: (1) the testis cell suspension was separated by sedimentation at unit gravity into “isolated cell” and “cell cluster” populations; (2) these populations were fractionated by isopyknic centrifugation in Percoll gradients. In terms of somatic cell composition, a nearly pure Sertoli cell (clusters) population was obtained between 1.017 and 1.033 g/ml and a Leydig cell (clusters) enriched population of between 1.033 and 1.048 g/ml (testes resuming spermatogenesis) or 1.048 and 1.062 g/ml (other testes).

These various cell populations were cultured in modified Leibovitz L15 medium for 10–15 days. When seeded, the Sertoli cells had a normal ultrastructure that remained unchanged for at least 10 days, and the steroidogenic activity of Leydig cells could be stimulated by salmon gonadotropin. Leydig cells remained 3β‐HSD positive and produced progesterone and 17α, 20β‐OH progesterone for at least 11 days.

This study points out that viable and differentiated trout somatic testicular cells can be prepared and cultured for several days.
Freezing and thawing alter chromatin stability of ejaculated human spermatozoa: Fluorescence acridine orange staining and Feulgen‐DNA cytophotometric studies
Authors: Royere D. , Hamamah S. , Nicolle J. C. , Barthelemy C. , Lansac J.
Published: February 2005

Cryopreservation and freezing‐thawing effects on the fertilizing ability of human spermatozoa commonly are evaluated by post‐thaw motility. Various studies have depicted the ultrastructural changes caused by freezing‐thawing, yet the chromatin alterations have been studied very limitedly. Our aim was to determine the extent to which freezing‐thawing alters the chromatin of human spermatozoa, using two analytical methods: acridine orange staining and Feulgen‐DNA cytophotometric studies. Both methods revealed a dramatic effect of freezing‐thawing on sperm chromatin: the native DNA content decreased as did the Feulgen‐DNA content, and sperm surface area was reduced. These results indicate an effect on DNA, diminished accessibility for Feulgen, and a decrease in nuclear surface area and prompt us to hypothesize a relationship between an “overcondensation” state for sperm chromatin after freezing‐thawing and a lower conception rate for human semen after cryostorage.
Zonae pellucidae of salt‐stored hamster and human eggs: Their penetrability by homologous and heterologous spermatozoa
Authors: Yoshimatsu Nobuhiro , Yanagimachi R. , Lopata Alex
Published: February 2005

Zonae pellucidae of unfertilized hamster and human eggs remain penetrable by homologous spermatozoa when they are stored in highly concentrated salt solutions [Yanagimachi et al., 1979]. Neutralization of salt solutions with the appropriate pH buffers is recommended for long‐term storage of the eggs (zonae), because zonae may gradually swell or even dissolve in acidic or alkaline solutions. At least for the hamster, 2 M ammonium sulfate solution containing 40 mM Hepes and 0.5% dextran (pH 7.0) seems to serve the purpose satisfactorily. Thorough rinsing of salt‐stored zonae prior to insemination is important, but inclusion of protease inhibitors in the rinsing medium does not appear to be essential. A dialysis method is recommended when a large number of salt‐stored eggs must be rinsed at one time. Salt‐stored hamster zonae, like fresh (control) zonae, permit attachment and penetration of hamster spermatozoa, but not human spermatozoa. Salt‐stored human zonae, on the other hand, allow penetration by not only human spermatozoa, but also hamster spermatozoa.
Does autocatalytic amplification of maturation‐promoting factor (MPF) exist in mammalian oocytes?
Authors: Fulka J. , Fléchon J.‐E. , Motlik J. , Fulka J.
Published: February 2005

The method of polyethylene‐glycol‐induced fusion of mammalian oocytes was applied to study maturation‐promoting factor (MPF) activity. After homologous fusions of one maturing–late diakinesis (LD), metaphase I (MI)–pig or mouse oocyte to one, two, or three immature–germinal vesicle (GV)–oocytes, giant cells were cultured in control or cycloheximide supplemented medium for 3 hours. The occurrence of germinal vesicle breakdown (GVBD) and premature chromosome condensation (PCC) served as a control of MPF activity. In giant cells composed of one maturing and one, two, or three immature oocytes, GVBD and PCC were observed in all cases after cultivation in the control medium. In the presence of cycloheximide, the completion of GVBD and PCC remained high when one maturing and one immature oocyte were fused (83.7% and 95.7% of GVBD in pig and mouse, respectively). However, in giant cells composed of one maturing and up to three immature oocytes, all GVs were broken down only occasionally (4.8% and 11.7% in pig and mouse, respectively). These results suggest that in pig and mouse oocytes MPF does not amplify autocatalytically, but requires active protein synthesis for its production.
Flow sorting of X and Y chromosome‐bearing mammalian sperm: Activation and pronuclear development of sorted bull, boar, and ram sperm microinjected into hamster oocytes
Authors: Johnson L. A. , Clarke R. N.
Published: February 2005

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome‐bearing bull, boar, and ram sperm populations and to separate the two sex‐determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 μM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X‐Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithio‐threitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P <.05). However, sorted and unsorted DTT‐exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P <.05). Treatment of sperm with DTT increased the activation rate (P < .05) for sorted boar sperm but not for bull or ram sperm. These data represent the first separation of bull, boar, and ram X and Y chromosome‐bearing sperm populations and the first evidence that sperm of domestic animals sorted on the basis of DNA by flow cytometric procedures have the ability to decondense and to form pronuclei upon injection into a hamster egg.
New mutation causing sterility in the mouse
Authors: Handel M. A. , Lane P. W. , Schroeder A. C. , Davisson M. T.
Published: February 2005

A new murine mutation, skeletal fusions with sterility, sks, has been identified. This mutation causes arrest during the pachytene stage of virtually all spermatogenic cells. Defects in chromosome pairing and appearance of the synaptonemal complex during meiosis in the male are apparent, but defective pairing is probably not the cause of sterility. Affected females are functionally infertile. Oocytes are capable of undergoing meiotic maturation in vitro but cannot be fertilized in vitro. Affected individuals of both sexes are characterized by fusions of vertebrae and of ribs. The sks gene has been mapped to Chromosome 4, 16.6 cM distal to the brown locus.
Oogenesis of the mayfly Habrophlebia eldae: Synthesis of vitelline and chorionic envelopes
Authors: Mazzini Massimo , Gaino Elda
Published: February 2005

The ultrastructure of developing ovarian follicles inside the panoistic ovarioles of Habrophlebia eldae were examined to observe the events occurring during egg maturation up to the full formation of the chorionic envelopes. The early vitellogenic follicles are coupled by gap junctions and are extensively interlocked with the oocyte plasma membrane via microvilli. With the onset of vitellogenesis, coated pits and coated vesicles are precursors to yolk deposition and are visible at the follicle cell‐oocyte interface. Postvitellogenic development entails the deposition of the egg envelopes. The vitelline envelope arises from the coalescence of rectangular plaques whose precursors are visible in Golgi complexes as heterogeneous electron‐opaque granules. A chorionic pattern of ridges on the egg surface characterizes the shell of H. eldae. The fully developed chorion shows three distinct regions with differently organized patterns. A fine layer of fibrous material (a secretion of the follicle cells, Ephemeroptera devoid of accessory glands) adheres to the egg chorion and is probably involved in attachment to the substrate.
Status of the rat acrosome during sperm‐zona pellucida interactions
Authors: Shalgi Ruth , Phillips David M. , Jones Roy
Published: February 2005

Over the past 40 years evidence from many sources has indicated that the mammalian acrosome reaction occurs within or near the cumulus oophorus. Recently, however, workers investigating in vitro fertilization in the mouse have concluded that in this system the acrosome reaction takes place on the surface of the zona pellucida. We have investigated the interaction of rat spermatozoa and the zona pellucida by using the scanning electron microscope (SEM) and two monoclonal antibodies which are directed to antigens of the rat sperm acrosome.

When in vitro inseminated eggs from which the cumulus has been removed are viewed with the SEM some sperm heads on the surface of the zona pellucida appear unaltered whereas others appear to be undergoing changes. In vivo, all displayed altered head morphology. Using immunogold labeling we found that the two antibodies employed, 2C4 and 5B1, were directed to acrosomal content and vesiculating acrosomal membranes. Immunofluoresence staining of zonae pellucidae in in vitro fertilization studies revealed numerous small positive regions. These were presumably acrosomal content and membranes which had been left on the zona surface by spermatozoa which had been associated with the zona surface. Our results suggest that the rat acrosome interacts with the zona pellucida. During this interaction some acrosomal content and membranes detach from the spermatozoon and remain on the surface of the zona pellucida.
Kinetics of spontaneous and induced acrosomal loss in human sperm incubated under capacitating and noncapacitating conditions
Authors: Byrd William , Tsu John , Wolf Don P.
Published: February 2005

The kinetics of spontaneous and induced acrosomal loss have been studied in human sperm incubated in capacitating and noncapacitating media. Acrosomal status was quantitated using indirect immunofluorescence with a monoclonal antibody. The response of sperm to induction by calcium ionophores was time dependent reaching a maximum after 6 hours of incubation under capacitating conditions. The inducible population slowly decreased in size through the balance of a 24‐hour incubation. The time‐dependent development of ionophore responsiveness by sperm exposed to capacitating conditions corroborates the idea that only capacitated cells can respond to undergo acrosomal loss in response to ionophore. In contrast, only a small, constant percentage of sperm incubated under noncapacitating conditions responded to ionophore. Substitution experiments involving the addition or deletion of human serum albumin suggest that albumin is not absolutely required for capacitation but is essential for the maintenance of motility. Polyvinyl alcohol can be substituted for serum albumin, but it does not support capacitation or motility as well as HSA. These studies may provide a basis for optimizing capacitating conditions for human sperm in vitro as well as for diagnosing fertility or fertility potential based on measurements of spontaneous and ionophore induced acrosomal loss under defined culture conditions.
In vitro capacitation and fertilizing ability of ejaculated rabbit sperm treated with lysophosphatidylcholine
Authors: Kim C. K. , Im K. S. , Zheng X. , Foote R. H.
Published: February 2005

Four experiments were replicated 1) to establish dose‐response relationships between lysophosphatidylcholine (LPC), sperm motility, and the acrosome reaction (AR), 2) to evaluate the influence of rabbit serum (RS) on these endpoints, 3) to compare buck differences in induction of the AR, and 4) to examine fertilizing ability in vitro of sperm tested under the first three objectives. Semen was collected from Dutch‐belted rabbits, washed once by centrifugation, resuspended, and preincubated for 2 or 4 hr in a chemically defined medium (DM), DM plus 20% RS, or BSA‐free DM plus 20% RS at 37°C. At the end of preincubation LPC was added to the preincubated sperm at concentrations of from 0 to 100 μg/ml. Sperm were examined .5–4 hr later for AR and sperm motility. For in vitro fertilization, sperm and ova were coincubated in DM up to 24 hr after insemination and in a more complex medium for another 24 hr. Addition of LPC to 4‐hr‐preincubated sperm was more effective for inducing the AR than addition to 2‐hr‐preincubated sperm. A significant increase (P < .05) in the AR occurred in 15 and 30 min following exposure to 100 and 80 μg of LPC per ml, respectively, but the higher concentration of LPC decreased sperm motility. Addition of 20% RS to DM with or without BSA surprisingly inhibited the AR but maintained sperm motility, as expected. Bucks differed (P < .05) in the initial percentage and the induced percentage of AR sperm. For the AR the optimal concentration of LPC per ml was 80 μg, but for in vitro fertilization 60 μg tended to be superior.
Effects of transglutaminase substrates and inhibitors on the motility of demembranated reactivated spermatozoa
Authors: de Lamirande Eve , Gagnon Claude
Published: February 2005

The effects of transglutaminase (TGase) substrates putrescine, dansylcadaverine, spermine, etc., and the TGase inhibitor cystamine were tested on the motility of demembranated mammalian spermatozoa. These products blocked within a few seconds the motility of demembranated reactivated spermatozoa at concentrations ranging from 0.25 to 5 mM. These minimal inhibitory concentrations could be decreased 5–150‐fold when TGase substrates and inhibitor were incubated with demembranated spermatozoa for 15 min prior to the addition of Mg·ATP. The inhibition was reversed by higher concentrations of Mg·ATP but none of these TGase substrates or inhibitor could inhibit bull sperm dynein ATPase. TGase activities, as measured by the incorporation of 3H‐putrescine into TCA‐precipitable proteins, were present in both sperm Triton‐soluble and ‐insoluble fractions. On the other hand, amine acceptor protein substrates for the TGase‐catalyzed reaction were present only in the insoluble fraction. The Triton‐soluble TGase was similar to the known “tissue” TGases; the Triton‐insoluble TGase activity was calcium independent. The same TGase substrates and inhibitor that blocked the motility of reactivated spermatozoa also blocked TGase activities. Linear relationships were observed between the concentrations of these substances required to block sperm motility and those to block TGase activities. These data suggest the involvement of a TGase activity in sperm motility.
Ultrastructural analysis of abnormalities in the morphology of the second meiotic spindle in ethanol‐induced parthenogenones
Authors: O'Neill G. T. , McDougall R. D. , Kaufman M. H.
Published: February 2005

A high frequency of parthenogenetic activation occurs when ovulated mouse oocytes are briefly exposed to a dilute solution of ethanol in vitro. Cytogenetic analyses of parthenogenones at metaphase of the first cleavage division have confirmed that parthenogenetic activation, per se, does not increase the incidence of chromosome segregation errors during the completion of the second meiotic division. Ethanol‐induced activation, however, significantly increases the incidence of aneuploidy. The ultrastructural changes that occur in the morphology and organization of the second meiotic spindle apparatus in ethanol‐ and hyaluronidase‐activated oocytes is reported here. Abnormalities in the arrangement of microtubule arrays and chromosome position were principally observed in ethanol‐activated oocytes at anaphase and telophase of the second meiotic division, but were only rarely observed in hyaluronidase‐activated oocytes. It is proposed that the abnormalities in spindle morphology and chromosome displacement observed in ethanol‐activated oocytes represent the initial events that lead to chromosome segregation errors following exposure to this agent.
Cytochemical study of intracellular calcium in hamster spermatozoa during the acrosome reaction
Authors: Ruknudin A.
Published: February 2005

Calcium was localized by a pyroantimonate technique in hamster spermatozoa during the acrosome reaction and pyroantimonate precipitates were observed in the anterior region of the acrosome. The calcium was also localized in the postacrosomal lamina of spermatozoa undergoing the acrosome reaction. Spermatozoa, incubated in capacitating medium containing verapamil, showed denser precipitates with an increase in concentration of this drug. Ionophore A23187 enhanced binding of calcium to the acrosomal region. The sodium channel inhibitor amiloride inhibited the acrosome reaction and the pyroantimonate precipitates were absent in these spermatozoa, whereas ionophore monensin enhanced the acrosome reaction. This suggests that the Na+/Ca++ antiporter may be responsible for intracellular Ca++ regulation during the acrosome reaction in hamster spermatozoa.
Proteins of the acrosomal region in mouse sperm: Immunological probes reveal post‐testicular modifications
Authors: Lakoski Katherine , Williams Carmen , Saling Patricia
Published: February 2005

Due to the central role the acrosomal region plays in sperm‐egg interactions, monoclonal antibodies (mAbs) were used to identify components of this domain in mouse sperm. Sev eral sperm proteins that localize specifically to the anterior acrosomal region are described here in terms of electrophoretic mobility, susceptibility to proteolytic degradation, and post‐translational modification during epididymal transit. Six different mAbs were used, each recognizing a distinctive antigen (Ag) or set of Ags in cauda epididymal mouse sperm: a doublet of 185/200 Kd (M42 mAb); 150‐160 Kd (M5 mAb); 105 Kd (W71 mAb); 21, 35, and 60 Kd (M41 mAb); 27 and 33 Kd (W33 mAb); and 57 and 86 Kd (W108 mAb). Previously reported work implicates two of these, M42 Ag and M5 Ag, as participants in sperm‐zona interaction (Saling and Lakoski: Biol Reprod 33:527‐536, 1985; Saling: Dev Biol 117:511‐519, 1986; and Lakoski et al.: Biol Reprod 38:221‐233, 1988).

Recognition of some (M42, M5, W108), but not all (W33), of the Ags by their corre sponding mAbs was affected by sperm incubation with proteases (trypsin or collagenase). Evidence of post‐translational modification during epididymal maturation was suggested by altered electrophoretic mobility of several of the Ags (M42, M5, W33, and W108) accompanying sperm transit from proximal to distal epididymis. Retention of sperm within the caput epididymis prevented structural alterations for the four proteins exam ined, indicating that spatial rather than temporal factors are critical for Ag modification in maturing mouse sperm.
Identification, isolation, and properties of a plasma membrane protein involved in the adhesion of boar sperm to the porcine zona pellucida
Authors: Peterson R. N. , Hunt W. P.
Published: February 2005

Boar sperm plasma membranes contain an integral protein (Mr 55 kDa) that apparently functions in the adhesion of sperm to the zona pellucida (Peterson and Hunt: J Cell Biol 105:170a, 1987.) In experiments described in this report, the protein is identified after additional steps of purification involving lectin affinity chromatography and preparative PAGE. An active form of the adhesion protein (APz) develops or becomes first exposed in the corpus epididymis and is fully active in the cauda epididymis; a significant portion of this conformationally labile protein, while integral to the plasma membrane, cannot be solubilized by nonionic detergents and may be associated with the membrane skeleton. APz does not exhibit enzymatic properties thought possibly to be involved in sperm‐zona interaction in this and other species. Galactosyltransferase substrates and inhibitors and anliproteases including soybean trypsin inhibitor, pepstatin, leupeptin, and p‐aminoben‐zamidine failed to block sperm from binding to porcine eggs. Boar sperm proacrosin and antiproacrosin antibody failed to inhibit sperm‐egg binding. When plasma membranes or fractions containing APz that bind to dextran sulfate agarose were chromatographed on L‐fucose agarose, a sugar which binds proacrosin, plasma membrane proteins that bound to the column failed to absorb anti‐APz antibody, Anti‐APz was absorbed by fractions that did not contain proacrosin. These data indicate that APz is not proacrosin. Since anti‐APz monovalcnt antibody raised from whole cauda or corpus sperm plasma membranes or from chromatographic fractions containing APz completely block capacitated sperm from binding to eggs, and since the ability of this antibody to be absorbed develops as sperm become capable of binding to eggs, we view AP, to be the major and perhaps only plasma membrane protein involved in the adhesion of capacitated boar sperm to eggs prior to the acrosome reaction.
A consistently successful procedure for in vitro fertilization of golden hamster eggs
Authors: Bavister Barry D.
Published: February 2005

Complete details are described for the first time of the procedures used in the author's laboratory for obtaining in vitro fertilization (IVF) of golden hamster eggs leading to the first cleavage division. These IVF procedures have been developed during the past 20 years and are very reproducible: IVF of at least 75% of eggs is routinely achieved, and on average 65% of inseminated eggs undergo the first cleavage division in vitro. These results can easily be obtained by inexperienced investigators. The ease and reproducibility of the hamster IVF procedures make them very suitable for studies of sperm:egg interaction and associated events. Studies in the author's laboratory have included analysis of sperm fertilizing ability under chemically defined conditions, the presence of sperm acrosome reaction stimulating factors in the egg investments, maturation of oocytes in vitro, the block to polyspermy, and the contribution of egg aging to fertilization anomalies. In addition, the motility of hamster sperm under chemically defined conditions is used in a routine screening protocol for detecting contaminants in the culture milieu. Golden hamster gametes of Ter several distinct advantages for IVF studies, including the large size of the sperm acrosome, the persistence of the very large sperm tail in the ooplasm for many hours following fertilization, and the translucence of the ooplasm, which facilitates observation of the sperm tail and pronuclei. The female golden hamster exhibits a regular 4 day estrous cycle, with distinctive indications of estrus and postestrus phases. Because of the advantages of using the golden hamster, the procedures described in this report may be useful to other investigators wishing to conduct research using IVF. Essentially the same IVF procedures can be used with monkey and bovine gametes.
Capacitation of hamster spermatozoa with the divalent cation chelators D‐penicillamine, L‐histidine, and L‐cysteine in a protein‐free culture medium
Authors: Andrews Jane C. , Bavister Barry D.
Published: February 2005

By using a chemically defined (protein‐free) culture medium that supports sperm viability but not capacitation or the acrosome reaction, we have determined that hamster spermatozoa can be chemically capacitated in vitro by the divalent cation chelators D‐penicilla‐mine, L‐histidine, and L‐cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1–2 × 106 sperm/ml) for 3, 4, or 6 hr at 37°C in 5% CO2 in air. The basic culture medium used for sperm preincubation and for sperm:egg coincubation was a modified Tyrode's solution (protein‐free) containing 10 mM sodium lactate, 100 μM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP‐PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 μM D‐penicillamine, 100 μM hypotaurine, and 1.0 μM epinephrine). The low control sperm preincubation medium consisted of TLP‐PVA. In some cases the high control preincubation medium also contained 3 mg/ml BSA (TALP‐PVA). The experimental preincubation medium was TLP‐PVA with additional D‐penicillamine (125 or 500 μM), or L‐histidine (10, 100, or 1,000 μM) or L‐cysteine (25, 75, or 125 μM). After preincubation, sperm were coincubated (2 × 104 sperm/ml) with cumulus‐free hamster eggs in TALP‐PVA ± additional D‐penicillamine, L‐histidine, or L‐cysteine for 1.5 hr, fixed, and evaluated for percent egg penetration as an index of sperm capacitation. The results demonstrate that hamster spermatozoa can be chemically capacitated in vitro with D‐penicillamine (500 μM: range of mean penetration values, 53.6%–84.3%), L‐histidine (100 μM: range of mean values, 24.8%–56.3%) or L‐cysteine (75 μM: 51.3%) in the absence of exogenous protein.
Morphogenesis of the decapitated and decaudated sperm defect in two brothers
Authors: Baccetti B. , Burrini A. G. , Collodel G. , Magnano A. R. , Piomboni P. , Renieri T. , Sensini C.
Published: February 2005

The “decapitated sperm” defect, found in both of two sterile brothers, may be assumed to have a genetic origin. The present material suggests that the term “decapitated spermatozoa” is not exact, because detached heads and tails were found in the brothers' ejaculate that could be regarded as “decapitated tails” and “decaudated heads.” The present report describes frequent, more or less advanced stages of detachment. Both heads and tails showed a normal structure in which only the postnuclear region was deficient, lacking basal plate and implantation fossa. A break at a different level of the midpiece, and therefore three kinds of separation, were observed. The defect, according to the present research, must originate in the testicular region, whereas the detachment occurs in the epididymis.
Fertilization and development of mouse eggs injected under the zona pellucida with single spermatozoa treated to induce the acrosome reaction
Authors: Lacham Orly , Trounson Alan , Holden Carol , Mann Jeff , Sathananthan Henry
Published: February 2005

Mouse spermatozoa were capacitated and acrosome reacted by incubation for 30 min to 6 h in modified Tyrode's medium (T6) and in cGMP. The fertilization rates of eggs microin‐jected with a spermatozoon from samples incubated for 30 min, 2 h, and 6 h in T6 and in cGMP were 36%, 34%, 29%, and 43%, respectively. These rates were not correlated to the percentage of acrosome‐reacted spermatozoa identified after these treatments. The lack of association could be explained by the selection of an increased proportion of acrosomc‐reacted spermatozoa actually used for microinjection, particularly in samples incubated for 30 min and 2 h in T6. Both acrosome‐intact and acrosome‐reacted spermatozoa were identified under the zona in the eggs which failed to fertilize. Fertilized eggs developed at very high rates to the blastocyst stage in vitro.
Maintenance of meiotic arrest in mouse oocytes by purines: Modulation of cAMP levels and cAMP phosphodiesterase activity
Authors: Downs Stephen M. , Daniel Susan A. J. , Bornslaeger Elayne A. , Hoppe Peter C. , Eppig John J.
Published: February 2005

Hypoxanthine and adcnosine are present in preparations of mouse ovarian follicular fluid, and these purines maintain mouse oocytes in meiotic arrest in vitro (Eppig et al.: Biology of Reproduction 33:1041–1049, 1985). The first hypothesis tested in this study is that purines which maintain meiotic arrest act by maintaining meiosis‐arresting levels of cyclic adenosine monophosphatc (cAMP) in the oocyte. Oocyte‐cumulus cell complexes were incubated in control medium (no added purines), or medium containing 0.75 mM adenosine, 4 mM hypoxanthine, or both for 3 hr and the percentage of the oocytes that underwent germinal vesicle breakdown (GVB) and the cAMP content of the intact complexes and the oocytes were determined. Adenosine alone had little inhibitory effect on GVB at this time point but sustained higher levels of cAMP in the oocytes. Hypoxanthine maintained 80% of cumulus cell‐enclosed oocytes in meiotic arrest and also sustained higher cAMP levels in the oocytes. The additon of adenosine to hypoxanthine‐containing medium increased the percentage of oocytes maintained in meiotic arrest, and increased the amount of cAMP in the oocytes above that maintained by either hypoxanthine or adenosine alone. Neither hypoxanthine, adenosine, nor hypoxanthine plus adenosine altered the cAMP content of intact complexes when assayed after 3 hr culture. Microinjection of an inhibitor of the catalytic subunit of cAMP‐dcpendent protein kinase induced GVB in denuded oocytes cultured in medium containing hypoxanthine. This purne, therefore, maintained meiotic arrest by sustaining elevated cAMP levels within the oocytes.

The second hypothesis tested in this study is that purines maintain meiosis‐arresting levels of cAMP, at least in part, by inhibiting cAMP phosphodiestcrase activity. In descending order of potency, 3‐isobutyl‐l‐methylxanthine (IBMX), guanosine, hypoxanthine, adenosine, and xanthosine inhibited cAMP phosphodiesterase in oocyte lysates. Moreover, like the potent phosphodiesterase inhibitor IBMX, hypoxanthine augmented the cAMP meiotic arrest and cAMP accumulation mediated by follicle‐stimulating hormone (FSH) in intact complexes. Therefore, inhibition of oocyte phosphodiesterase appears to be one mechanism by which the purines could maintain meiosis‐arresting levels of cAMP.
Effects of semen preservation on boar spermatozoa head membranes
Authors: Buhr M. M. , Canvin A. T. , Bailey J. L.
Published: February 2005

Head plasma membranes were isolated from the sperm‐rich fraction of boar semen and from sperm‐rich semen that had been subjected to three commercial preservation processes: Ex tended for fresh insemination (extended), prepared for freezing but not frozen (cooled), and stored frozen for 3‐5 weeks (frozen‐thawed). Fluorescence polarization was used to determine fluidity of the membranes of all samples for 160 min at 25°C and also for membranes from the sperm‐rich and extended semen during cooling and reheating (25 to 5 to 40°C, 0.4°C/min). Head plasma membranes from extended semen were initially more fluid than from other sources (P < 0.05). Fluidity of head membranes from all sources decreased at 25°C, but the rate of decrease was significantly lower for membranes from cooled and lower again for membranes from frozen‐thawed semen. Cooling to 5°C reduced the rate of fluidity change for plasma membranes from the spernvrich fraction, while heating over 30°C caused a signifi cantly greater decrease. The presence of Ca++ (10 mM) lowered the fluidity of the head plasma membranes from sperm‐rich and extended semen over time at 25°C but did not affect the membranes from the cooled or frozen‐thawed semen. The change in head plasma membrane fluidity at 25°C may reflect the dynamic nature of spermatozoa membranes prior to fertilization. Extenders, preservation processes and temperature changes have a strong influence on head plasma membrane fluidity and therefore the molecular organization of this membrane.
Ultrastructural and histochemical changes in the murine zona pellucida during the final stages of oocyte maturation prior to ovulation
Authors: Kaufman M. H. , Fowler R. E. , Barratt E. , McDougall R. D.
Published: February 2005

The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi‐thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra‐thin sections were examined by transmission electron microscopy. A parallel series of semi‐thin sections of non‐osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm‐zona binding and sperm‐egg interactions.