bluuebunny/crossref_metadata_2025 huggingface dataset:
The generation and propagation of double waves in mosquito (Aedes notoscriptus) sperm‐tails
Authors: Swan M. A.
Published: February 2005

In the mosquito Aedes notoscriptus nine accessory tubules develop from the doublets of the axoneme whose single central fibre forms late in spermiogenesis. The 9 + 9 + 1 mosquito sperm‐tail propagates two waves of differing frequency and amplitude. The structural basis for the movement is of interest.

Mosquito sperm were demembranated until all movement ceased and were reactivated with a solution containing adenosine triphosphate (ATP). They immediately moved in a normal fashion. Like untreated sperm, they always propagated double waves. This indicates that the plasma membrane does not control the double wave.

After sonicating sperm treated as above, the high‐frequency, low‐amplitude waves sometimes propagated alone beyond a damaged region of sperm‐tail, whereas the large‐amplitude, low‐frequency waves never propagated alone beyond a damaged region. This suggests that the latter waves are generated by an anterior site, possibly the centriole or centriole adjunct. The structure that propagates the large amplitude wave is not known; possibly the accessory tubules may slide against the axonemal doublets.

Any region of the sperm‐tail is capable of generating and propagating the low‐amplitude, high‐frequency wave, for which the structure responsible is probably the axoneme.
Immunoferritin labeling of rabbit spermatozoa
Authors: Schrader Steven M. , Eng Ludeman A. , Metz Charles B.
Published: February 2005

Ferritin‐conjugated goat IgG binds nonspecifically to rabbit sperm. This restricts use of ferritin‐labeled goat antiglobulins as indirect labels in rabbit sperm antigen localization. Ferritin‐conjugated Protein A does not bind nonspecifically to rabbit sperm and is a satisfactory substitute for indirect (“secondary”) labeling of sperm antigens.
cAMP‐dependent protein kinase in sea urchin embryos
Authors: Fujino Yukio , Yasumasu Ikuo
Published: February 2005

cAMP‐dependent protein kinase in the supernatant fraction of the homogenate of sea urchin eggs and embryos obtained by centrifugation at 105,000g was investigated in the present study. In the previous report, the dissociation constant between cAMP‐binding proteins and cAMP changed during the development. This suggests that the nature of cAMP‐dependent protein kinase, which has been well established to be the major cAMP receptor, changes during the development. In the present study, four protein kinases were separated through DEAE‐cellulose column from the supernatant of unfertilized egg homogenate. One of them was cAMP‐dependent protein kinase. The others were cAMP‐independent ones. One among them was phosvitin kinase, and the others were not identified at present. The activity of cAMP‐dependent protein kinase gradually increased during a period from fertilization to the swimming blastula stage. During this period, cleavages occurred at a high rate, and the rate decreased after hatching out. Thus, it is supposed that cAMP‐dependent protein kinase in the supernatant may take a part in the mechanism of cleavage. The activity, however, became very low at the mesenchyme blastula, the gastrula, and the pluteus stages. cAMP‐binding capacity was observed in the sedimentable fraction and the supernatant fraction, respectively, obtained by 105,000g centrifugation at all stages examined. If the structure‐bound cAMP‐binding protein is also cAMP‐dependent protein kinase, it may play different roles in the mechanism of development.
Isolation and characterization of two proteinase inhibitors from the male reproductive tract of mice
Authors: Poirier Gary R. , Jackson James
Published: February 2005

Low molecular weight, acid‐stable proteinase inhibitors from epididymal and seminal vesicle homogenates were isolated and characterized. The isolation procedure consisted of gel filtration, trypsin affinity, and ion exchange chromatography. The inhibitor from seminal vesicle homogenates has a molecular weight of approximately 6,200, and that of the epididymal inhibitor was estimated at 4,000. Antiserum directed against the seminal vesicle inhibitor did not react with epididymal components. The epididymal inhibitor shows competitive, whereas the seminal vesicle inhibitor shows noncompetitive inhibition against trypsin on double reciprocal plots. Both inhibitors are effective against trypsin and acrosin but not against chymotrypsin, kallikrein, thrombin, or plasmin. To verify site of origin and to investigate androgen dependency of the epididymal inhibitor, mice were efferentiectomized, orchiectomized, or orchiectomized with androgen supplementation. Gel filtration profiles of acid‐treated epididymal homogenates from normal and efferentiectomized animals show inhibitor peaks in the same regions. The concentration of acid‐stable inhibitor from epididymal homogenates decreased with orchiectomy but returned to normal values when exogenous androgen was supplied. These observations suggest that the low molecular weight inhibitor in the epididymal homogenates is distinct from that in the seminal vesicles. Furthermore, the inhibitor associated with epididymal homogenates is androgen‐dependent, and the epididymis is the site of origin of this inhibitor.
The effect of nicotine on sperm‐Egg interaction in the sea urchin: Polyspermy and electrical events
Authors: Dale B. , de Santis A. , Hagström B. E.
Published: February 2005

We have studied some of the effects of nicotine on sea urchin eggs, spermatozoa, and their interaction using electrical recording techniques and fertilization‐rate experiments. Pretreating eggs with nicotine enhances the fertilization rate, whereas this drug has an inhibitory effect on spermatozoa. Pulse‐treated eggs or eggs fertilized in the presence of nicotine give rise to attenuated step depolarizations, which may be attributed to a decrease in membrane resistance (Rm) of the egg or, in the latter case, to an alteration to the spermatozoon. Concurrently, with the change in the step depolarization there is a reduction in amplitude of the fertilization potential (FP) suggesting that the cortical reaction is in some way altered. Nicotine has no effect on the Rm of fertilized eggs or oocytes, where there are no cortical granules. We suggest that nicotine alters the cortex of sea urchin eggs–possibly by causing a partial dissolution of cortical granules–which renders the eggs more receptive to spermatozoa. The reductions in amplitude of the step depolarization and the FP are consequences of this alteration.
Some nucleocytoplasmic exchanges during spermiogenesis of boar, ram, and stallion
Authors: Courtens J. L.
Published: February 2005

The problem of how the successive nucleoproteins enter and leave the nucleus of mammalian spermatids is studied with electron microscopy in thick and thin sections of testis, stained en bloc with the procedure of Thiery and Rambourg [1976], which is able to immobilize small molecules. Staining at different pH values reveals that the stain could demonstrate some spermatidspecific nucleoproteins in elongating nuclei and the spermatozoa‐specific protamines in elongated nuclei of the boar, the ram, and the stallion. The stained substances enter or leave the nuclei at precise steps in spermiogenesis. They follow several ways inside a special apparatus made rigid with the manchette. The apparatus is composed of the endoplasmic reticulum, continuous with the nuclear envelope, and of the nuclear pockets continuous with the nuclear pores. In the stallion and the boar, cytoplasmic granules, surrounded by a double wall of membranes, fuse with the nuclear envelope at the time the protamines enter the nucleus.
Ultrastructural observations on cortical granules in human follicular oocytes cultured in vitro
Authors: Sathananthan A. H. , Trounson A. O.
Published: February 2005

The fine structure, distribution, and fate of cortical granules in human oocytes cultured in vitro are reported.

Follicular maturation in women with blocked Fallopian tubes was induced by clomiphene citrate and human chorionic gonadotropin, and preovulatory eggs were obtained by improved methods of laproscopy and oocyte recovery. These oocytes were then inseminated and cultured in a modified Ham's F10 medium for 3 to 72 hr to assess their fertilizability.

Cortical granules were observed in all 17 unfertilized oocytes investigated, which had completed various stages of meiotic maturation. A marked increase in their numbers was observed in oocytes cultured for 3 to 6 hr. There was no evidence of spontaneous cortical granule release in any of the oocytes studied.

It is concluded that cortical maturation expressed by proliferation of cortical granules is as significant a criterion as nuclear maturation in assessing maturity and fertilizability of oocytes cultured in vitro. A short sojourn in culture before insemination could improve chances of normal fertilization and embryo development, which has been recently achieved in our laboratory.
Steroidogenesis by isolated porcine oocyte‐cumulus complexes: Lack of an inhibitory effect of low molecular weight fraction of porcine follicular fluid on conversion of androgen to estrogen
Authors: Schaerf Frederick W. , Anderson Larry D. , Channing Cornelia P.
Published: February 2005

The ability of isolated porcine oocyte‐cumulus complexes to secrete progesterone and convert androgens to estrogen during two days of culture was examined. We studied the effects of steroids, as well as a partially purified fraction of follicular fluid oocyte maturation inhibitor (Sephadex Peak A OMI), on the ability of oocyte‐cumulus complexes to mature and convert androgen to estrogen.

The addition of 0.014, 0.14 or 1.4 μg/ml androstenedione to the culture medium resulted in a substrate dose‐dependent accumulation of estrogen in the culture medium after two days. Oocyte‐cumulus cell complexes secreted more estrogen in the presence of androstenedione than in the presence of testosterone (P < 0.05). The addition of 1.4 μg/ml testosterone, androstenedione, or estradiol, but not dihydrotestosterone, inhibited cumulus cell progesterone secretion (P < 0.001 versus untreated control culture). Oocyte maturation was not altered by the addition of steroids in doses up to and including 1.4 μg/ml. The Sephadex Peak A OMI fraction of pFFL inhibited oocyte maturation 51% (P < 0.01) and progesterone secretion 91% (P < 0.01) but had no effect on the conversion of androgens to estrogens. Cumulus cell monolayer formation was inhibited 71.5% (P < 0.01) by the Sephadex Peak A OMI fraction and 35.4% (P < 0.05) by the Sephadex Peak A OMI fraction plus androstenedione.

These studies indicate that porcine oocyte‐cumulus complexes can convert androgens to estrogens and that partially purified OMI does not inhibit conversion of androgens to estrogen.
Sperm‐egg ratios and the site of the acrosome reaction during in vivo fertilization in the hamster
Authors: Cummins J. M. , Yanagimachi R.
Published: February 2005

Little is known about the timing of the mammalian sperm acrosome reaction during fertilization in vivo. To study this problem, female hamsters were inseminated at about the time of ovulation, and the contents of the ampullary regions of their oviducts were subsequently examined at various intervals. No living spermatozoa were recovered from ampullae earlier than 4 hr after insemination. The first appearance of living spermatozoa coincided closely with the first appearance of fertilized eggs in the same oviduct. The total numbers of living spermatozoa did not start to exceed the number of eggs in the same ampulla, until after 50% or more of the eggs had been fertilized. Hamster spermatozoa are highly efficient at making contact with eggs, and the fertilizing spermatozoon probably spends no more than 2½ –5½ min in penetrating the cumulus oophorus. Spermatozoa that enter the ampulla appear to be ready to undergo the acrosome reaction, and complete it while they are passing through the cumulus or shortly before, or after, contacting the surface of the zona pellucida.
Use of a radioimmunoassay technique to measure the titer of anti‐cow‐zona antibodies in marmoset monkeys
Authors: Hulme M. J. , Aitken R. J. , Richardson D. W.
Published: February 2005

A double antibody radioimmunoassay technique is described for the measurement of anti‐zona antibodies in the peripheral plasma of marmosets actively immunized against intact cow zonae pellucidae. The method has been shown to be a reliable, repeatable indicator of antibody titer, enabling direct comparison of the response obtained between marmosets. This is the first report of a procedure describing the active immunization of a primate against zona antigens, and the first time a radioimmunoassay technique has been used to monitor profiles of anti‐cow zona antibody production following active immunization. The method should prove to be a useful tool in the evaluation of zona antigens as agents for immunocontraception.
Circular DNA in human and boar spermatozoa
Authors: McGrath J. P. , Evenson D. P.
Published: February 2005

Circular DNA molecules were isolated from human and boar whole spermatozoa or spermatozoal nuclei and measured for size by electron microscopy. The DNA molecules derived from both mammals were heterogeneous in size ranging from 0.07 to 17 μm; nearly 75% of the molecules were ⩽0.5 μm in length. The mean lengths were 1.0 μm and 1.5 μm for circular DNAs isolated from human and boar spermatozoa, respectively. The origin and function of these molecules remains unknown.
Change in intracellular distribution of cAMP‐binding capacity in sea urchin embryos during early development
Authors: Fujino Yukio , Yasumasu Ikuo
Published: February 2005

In the sea urchin, Strongylocentrotus intermedius, cAMP‐binding capacity was found in the sedimentable fractions obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min and 105,000g for 1 h, and also the supernatant fraction by centrifugation at 105,000g. The cAMP‐binding capacity was estimated depending on the radioactivity of 3H‐cAMP found in the nonfiltered matter obtained by a filtration through a Millipore filter of a mixture containing 3H‐cAMP and each fraction. Prior to the filtration, the mixture was incubated at 0°C for 1 hr. In all fractions, the addition of nonlabeled cAMP to the mixture containing 3H‐cAMP resulted in a marked decrease in the radioactivity in the nonfiltered matter. cGMP, cTMP, and cUMP caused only a slight decrease in the radioactivity in the nonfiltered matter. These indicate that specific cAMP binding occurs in the nonfiltered matter of all fractions. The extent of decrease in 3H‐radioactivity by cIMP was higher than that caused by cAMP in the 10,000g sedimentable fraction, but in the other fractions, it was as low as observed when cGMP, cTMP, and cUMP were added. This suggests that the nature of cAMP‐binding matter in this fraction differs somewhat from those in the other fractions. cAMP‐binding capacity in this fraction (10,000g sedimentable fraction) increased gradually during development and the capacity in the other fractions, especially in the supernatant fraction, decreased at the gastrula stage. Protein kinase activity was also found in these fractions. Alteration of intracellular distribution of cAMP‐binding proteins and protein kinases during development probably shows the change in the role of protein kinase in the mechanism of development.
Novel procedures for collection of sea urchin egg cortical granule exudate: Partial characterization and evidence for postsecretion processing
Authors: Baginski Richard M. , McBlaine Paul J. , Carroll Edward J.
Published: February 2005

We have developed two procedures to collect total cortical granule exudate in a soluble form from eggs of the sea urchin Strongylocentrotus purpuratus. Egg suspensions were either treated with dithiothreitol to disrupt the vitelline envelope or divalent cations were removed postinsemination to prevent the normal vitelline‐to‐fertilization envelope transition. Rapid acidification of the insemination mixture (dithiothreitol‐treated eggs) to pH 6.0 prevented precipitation of the paracrystalline protein fraction described by Bryan [1970a]. Exudate was partitioned into three fractions. The pH 8.0‐insoluble fraction appeared to be identical to the paracrystalline protein fraction. The pH 8.0‐soluble fraction was separated into pH 4.0‐soluble and‐insoluble fractions. Analysis for peroxidase and protease activities showed that peroxidase activity was localized in all three fractions whereas protease activity was restricted to the pH 4.0 insoluble fraction as reported [Carroll and Epel, 1975]. A minimum of six major proteins were detected on native polyacrylamide gels of total exudate. Under reducing and denaturing conditions, 12 polypeptides ranging from 19,000 to 165,000 in molecular weight were detected in total exudate; six polypeptides were recovered in the pH 8.0‐insoluble fraction. To test the hypothesis that protease and peroxidase activities process cortical granule proteins after secretion, we inseminated eggs in solutions containing peroxidase and protease inhibitors. The paracrystalline protein fraction crystallized slowly from insemination mixtures containing both inhibitors compared to controls and there were dramatic differences in exudate electrophoretic patterns. We suggest that cortical granule protease and peroxidase activities process the exudate so that the paracrystalline protein fraction rapidly crystallizes during normal fertilization.
Hyperactivated motility patterns of ram spermatozoa recovered from the oviducts of mated ewes
Authors: Cummins J. M.
Published: February 2005

The contents of the oviducts of ewes were recovered by flushing with small volumes of culture medium, 22½–24¼ hr after mating. The ampulla was flushed separately from the uterotubal junction and isthmus. Among the motile spermatozoa recovered, a proportion exhibited “hyperactivated” motility, also known as “activated”, or “whiplash” motility. This was characterized by increased flexion of the neck, increased amplitude of the flagellar waves, and marked asymmetry of beat. Two types of hyperactivation appeared: in the first, spermatozoa swam in a repetitive, nonprogressive circling pattern and appeared to have intact acrosome caps; in the second, the spermatozoa showed a propensity to stick to glass by the equatorial segment and most had modified or missing acrosome caps. The proportions of motile spermatozoa exhibiting hyperactivation were greatest in the ampullae, as were the proportions with modified or absent acrosomes. Hyperactivation is a capacitation‐associated phenomenon that has now been reported for one or more species from seven orders of eutherian mammals. It may well be a universal aspect of the prefertilization behavior of mammalian spermatozoa and is probably of advantage to the fertilizing spermatozoon within the oviduct.
Activities of X‐linked enzymes in spermatocytes of mice rendered sterile by chromosomal alterations
Authors: Hotta Y. , Chandley A. C.
Published: February 2005

Spermatocytes in the late prophase of first meiotic division isolated from sterile males retain higher activities for three X‐linked enzyme5, phosphoglycerate kinase (PGK)‐1, glucose‐6‐phosphate dehydrogenase (G6PD), and hypoxanthine‐guanine phosphoribosyl transferase (HGPRT) than those of fertile males. The sterilty of the male is presumed to be owing to the rearrangement of X‐chromosome material and the possibility of abnormal meiotic X‐chromosome inactivation is discussed.
Effect of antibody to detergent solubilized zonae pellucidae on in vivo and in vitro fertilization in the mouse
Authors: Tsunoda Y. , Sugie T.
Published: February 2005

An antiserum was produced in one rabbit against mouse zonae pellucidae solubilized with 70 mM Na2SO3, 1% SDS, and 0.04 mM CuSO4. An IgG of zona antibody completely inhibited fertilization both in vitro and in vivo in the mouse. F(ab′)2 fragment obtained by pepsin digestion of IgG zona antibody inhibited fertilizability of eggs in vitro but did not inhibit fertilization in vivo after passive immunization.
Calcium and adenosine affect human sperm adenylate cyclase activity
Authors: Hyne Ross V. , Lopata Alex
Published: February 2005

The adenylate cyclase activity of human ejaculated spermatozoa in broken‐cell preparations was investigated. In the presence of 5 mM metal cations and 0.1 mM ATP, the relative enzyme activity with Mn2+, Ca2+, Mg2+, Ba2+ was 1.00, 0.28, 0.22, and 0.03, respectively. Added Ca2+ appeared to activate the enzyme in the presence of Mn2+ or Mg2+. The human sperm adenylate cyclase was stimulated by ∼ 2‐fold by free Ca2+ (lmM) in the presence of Mg2+ (5 mM). If the GTP analogue, 5′‐guanylyl imidophosphate (Gpp(NH)p) was added to the sperm homogenate in the presence of 200 μM ethylene‐glycol‐bis (β‐aminoethylether) N,N′‐tetraacetic acid (EGTA), the adenylate cyclase activity was increased by approximately 25%, but with the addition of 280 μM Ca2+ there was a decrease in enzyme activity. A similar response to low concentrations of Ca2+ was obtained after complementation of the sperm enzyme with the guanine nucleotide regulatory component from human erythrocytes, where the addition of 40 μM Gpp(NH)p, 200 μM EGTA, and Ca2+ (≤ 160 μM) stimulated the sperm enzyme ∼ 3–4‐fold, but the further addition of Ca2+ (280 μM, final) neutralized the stimulatory effect. The addition of adenosine, and the nucleotides 5′‐AMP and 5′‐ADP inhibited the enzyme, whereas guanine and 5′‐GMP had no appreciable effect. Human follicular fluid and serum also had little direct effect on the sperm adenylate cyclase. These resuls suggest that Ca2+ might be an important physiological modulator of the human sperm adenylate cyclase.
Sperm motility in the horseshoe crab. III. Isolation and characterization of a sperm motility initiating peptide
Authors: Clapper David L. , Epel David
Published: February 2005

Sperm motility in Limulus is initiated by a sperm motility initiating factor (SMI) that emanates from Limulus eggs. This report describes the partial purification of SMI (greater than 230‐fold purification with respect to protein content) with 40% recovery. SMI appears to be a hydrophobic peptide of 500–2,000 MW. Although probably not purified to homogeneity, SMI is estimated to be active at a concentration of less than 0.2 μM.
Effect of an extract of nereis virens on mammalian spermatozoa
Authors: Gordon Mildred , Morris Eugene G. , Stark Freddy , Williams William L. , Young Ron J.
Published: February 2005

Epididymal sperm of the mouse, rat, and guinea pig and ejaculated sperm of rabbits are cleaved at the head‐tail junction by an extract of Nereis virens. Annelids are extracted with water and the extract is purified by ion exchange chromatography. Electron microscopy shows that the extract acts on the filaments connecting the capitulum of the tail with the basal plate lining the nuclear envelope. Following detachment, the basal plate remains with the head. The extract contains proteases as indicated by hydrolysis of tosyl arginine methyl ester (TAME), benzoyl arginine ethyl ester (BAEE), and Azocoll, a general protease substrate. The hydrolysis of TAME is inhibited by tosyl lysine chloromethyl ketone (TLCK), a trypsin inhibitor, but TLCK does not prevent head‐tail separation by the Nereis extract. Similarly tosyl phenylalanine chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and phosphoramidon and leucyltryptophan, both thermolysin and acrolysin inhibitors — singly or in combination — do not prevent hydrolysis of Azocoll. Head‐tail separation activity of the extract was inhibited by dithiothreitol, which reduces disulfide bonds, and phenylmethyl sulfonyl fluoride, an inhibitor of serine proteases. These results indicate that the extract is a mixture of proteases, one being a serine protease similar to trypsin.

Digestion of the connecting filaments with the pure proteases, trypsin and Staphylococcus aureus V8 protease, has yielded the following information on the proteins of the filaments. The accessibility of arginine and/or lysine peptide bonds to enzyme action is highest in rat sperm filaments, whereas those in the filaments of mouse, rabbit, and guinea pig sperm are less accessible than in the rat. Another possibility is that the total content of arginine and/or lysine varies between the species. The most dramatic difference is the enzymatic action on glutamyl peptide bonds of the filaments, the order being: mouse 〉 rat 〉 rabbit, with guinea pig sperm filaments completely resistant over the time course of the experiment.
Stabilization in vivo and in vitro of the spermatozoon head and tail to detachment induced by primary amine, thiol, and detergent
Authors: Young R. J. , Cooper G. W.
Published: February 2005

Rabbit spermatozoa recovered from the reproductive tract of females 12–13 hr postcoitum resisted head/tail separation induced by n‐butylamine and dithiothreitol, but not sodium dodecylsulfate. Stabilization of the sperm head/tail junction also developed in vitro at 37°C in physiological media and in saline‐Tris‐HCl (pH 7.4). Resistance to dithiothreitol occurred in motile, but not immotile spermatozoa. Only nonmotile spermatozoa developed resistance to sodium dodecylsulfate in vitro, whereas both motile and immotile spermatozoa became resistant to n‐butylamine. Stabilization to n‐butylamine was time and temperature dependent and was accelerated by Cu2+, Mg2+, and Zn2+, but not Mn2+. The resistance of hamster and rabbit spermatozoa to n‐butylamine developed in physiological media over the same time intervals as required for capacitation and the acquisition of hyperactivated motility.

Reagents that react with sulfhydryl groups had no effect on the development of resistance to n‐butylamine but inhibited stabilization to sodium dodecylsulfate, suggesting that the latter stabilization may result from the formation of disulfide crosslinks at the head/tail junction. Reduction of aldehyde groups by sodium cyanoborohydride did not prevent stabilization to sodium dodecylsulfate, but did reduce detachment by dithiothreitol. Aldehyde groups thus are not involved in the stabilization of the head/tail junction to sodium dodecylsulfate, but may participate in new crosslinks stabilizing the head/tail junction to dithiothreitol. Inhibitors of transglutaminase did not prevent development of resistance to n‐butylamine, sodium dodecylsulfate, or dithiothreitol indicating that head/tail stabilization does not involve intermolecular γ‐glutamyl‐ϵ‐dysine bonds.
Temporal synthesis of mitochondrial proteins from mouse epididymal spermatozoa
Authors: Hecht Norman B. , Kennington Ellzabeth
Published: February 2005

Mitochondria from ejaculated bovine spermatozoa contain a group of polypeptides ranging in molecular weights from 13,000 to 35,000 not found in other bovine or murine testicular mitochondria [Hecht and Bradley, 1981]. These proteins are present in the mitochondria isolated from both epididymal and ejaculated spermatozoa. To establish when during epididymal transport, spermiogenesis, and/or meiosis these proteins are synthesized, the synthesis intervals for the mitochondrial proteins from cauda epididymal spermatozoa were established following intratesticular injection of (35S)methionine. Mice were killed every third day over a 33‐day period and cauda epididymal spermatozoa were fractionated into mitochondrial and head components. Radioactivity in each fraction was monitored by liquid scintillation counting. Maximal incorporation was observed during spermiogenesis, although substantial amounts of protein were synthesized during meiosis. Analysis of the mitochondrial polypeptides by gel electrophoresis revealed that many polypeptides such as the cysteine‐rich structural protein of the mitochondrial capsule were synthesized over prolonged intervals of spermiogenesis and meiosis rather than in a brief specific time period. These results suggest that spermatozoal mitochondria are produced by a sequential substitution of new proteins into the differentiating mitochondria rather than the abrupt appearance of a new class of mitochondria during spermatogenesis.
Active immunization of cynomolgus monkeys (Macaca fascicularis) with porcine zonae pellucidae
Authors: Gulyas Bela J. , Gwatkin Ralph B. L. , Yuan Lydia C.
Published: February 2005

Cynomolgus monkeys (Macaca fascicularis) were immunized with porcine zonae pellucidae to assess the possible antifertility effects of the zona antibodies. Serum antibody titers were evaluated utilizing a rapid solid‐phase radioimmunoassay. Six of twelve monkeys conceived 6 to 10 wk after vaccination. All monkeys reached maximal antiserum titers by the time of conception, although the six animals that did not conceive had considerably lower antibody titers. Further pregnancies did not occur until antibody level had declined markedly, 8 mo after last immunization. The menses of all but one of the remaining six monkeys were interrupted intermittently. Also, the usual midcycle elevated estradiol levels were absent for several cycles. Both menses and midcycle estradiol peaks were reestablished in all but one monkey 3 to 5 mo after the last booster was given. Two monkeys conceived when serum antibody levels dropped to one fourth of maximal, but both had a still birth. Histological observations showed accumulation of luteal tissue and massive atresia of small follicles at the end of the study (18 mo). We conclude that through heteroimmunization with porcine zona pellucida monkeys can become infertile and that this condition is reversible. Because the zona preparation used in this study appeared to contain traces of nonzona material, it was not possible to determine whether the menstrual irregularities and oocyte atresia that we observed were owing to immunological effects on the zona itself or to the production of antibodies against other ovarian components.
Effects of egg aging on in vitro fertilization and first cleavage division in the hamster
Authors: Juetten Jacquline , Bavister Barry D.
Published: February 2005

The postovulatory fertile life of mammalian eggs is remarkably short (approximately 6‐36h). Anomalies of embryogenesis may result from fertilization of aged, defective eggs. Attempts to study this problem using whole‐animal models are complicated by chances in the natural milieu of the gametes. In the present study, postovulatory hamster eggs were allowed to agein vivo then fertilized in vitro. Cumulus‐intact eggs recovered from superovulated hamsters either 2 or 9 h after the estiated time of ovulation (12 h postHCG) were incubated for 4 h with preincubated sperm suspentions in a modified Tyrode's solution devised for in vitro fertilization. Eggs were either fixed or cultured for another 20h in fresh medium to allow cleavage to occur, then examined by light microscopy (phase and interference‐contrast). No significant difference was found in the ablities of fresh and aged eggs to be penetrated by spermatozoa (94% vs 90%, respectively; 8 replicated experiments), but only 59% of penetrated aged eggs were found to undergo morphologically normally fertilization (2 polar bodies, 2 prounclei) compared with 75% of fresh eggs (difference significant, P< 0.01). About 13% of eggs were polyspermic in both categories. The most common anomaly in aged fertilied egges was failure to extrude the second polor body (23% off eggs vs 8% of fresh eggs, P < 0.01). Only 21% of aged eggs underwent first cleavaage, and only 74% of these appeared morphologically normal, compared with value of 68% and 98%, respectively, for fresh eggs. These data show that in the hamster, abnormal fertilization and cleavage failure can, in part, be directly attributed to postovulatory deterioration of eggs. We also infer that the apparently very short penetrable life of hamster eggs in vivo shown by previous investigators is an indirect effect of postovulatory changes in the female reproductive tract that are unfavorable for sperm‐egg interactions.
A species‐specific sperm factor dispersing the jelly coat of the egg of the sea urchin, Anthocidaris crassispina
Authors: Yamada Yuji , Aketa Kenji
Published: February 2005

A species‐specific factor capable of disersing the jelly coat surrounding eggs has been purified from sperm of the sea urchin, Anthocidaris crassisina. It does not exert its effect on the vitelline layer. The purification has been accomlished by a four‐step procedure involving ammonium sulfate fractionation, gel filtration on Sepharose CL‐4B, ion‐exchange column chromatography on DEAE‐cellulose, and affinity column chromatograhy on heparin‐Seharose CL‐6B. The isolated factor is homogenous in sodium dodecyl sulfate polyacrylamide gel electrohoresis in the presence or absence of β‐mercatoethanol, estimated molecular weight being about 140,000.

The jelly dispersion by the present factor is activated by CaCl2, and inhibited by KCl, MnCl2, EDTA, and EGTA, and by sulfated saccharides such as chondroitin sulfate A and C, heparin, and glucose‐6‐sulfate, Inorganic sulfated such as (NH4)2SO4 and Na2SO4 have no effect on jelly dispersion. This factor is heat‐labile, its activity in 30 min at 50°C.

The present factor is found also in the seminal Plasma, and released from sperm themselves by treatment with Triton X‐100 .These results suggest that this factor is loosely bound to the serm surface. Although glycosidase and arylsulfatase activities are detectable in the seminal plasma, these enzyme activities are not detectable in the purified jelly disersing factor.

Only trypsin and α chymotrysin among commercial enzymes tested dispersing activity is inhibited neither by trypsin inhibitors such as N‐α‐p‐tosyl‐L‐lysine‐chloromethyl ketone, soybean trypsin inhibitor, ovomucoid trypsin inhibitor, nor by chymotrypsin inhibitors such as L‐1‐tosylamide‐2 pheny‐ethylcholoromethyl ketone and chymostatin Participation of trysin‐like and chymotrypsin‐like enzymes in jelly dispersion seems unlikely.
Ultrastructure of a crystalline inclusion in the cytoplasmic droplet of bovine spermatozoa
Authors: Olson G. E. , Winfrey V. P. , Noland T. D. , Garbers D. L.
Published: February 2005

An elongate crystalloid inclusion has been noted in the cytoplasmic droplet of cauda epididymal bovine spermatozoa. The crystalloid appears to be composed of an aggregate of parallel 10 nm diameter filamentous elements that are associated laterally with one another. It has a regular cross‐banding pattern that repeats at 13–15‐nm intervals. A purified fraction of detached droplets was prepared by centrifugation of sperm suspensions onto Percoll gradients. The detached droplets also exhibited the crystalline inclusion. The origin and possible functions of this structure are discussed.
On the role of glucose in capacitation and acrosomal reaction of guinea pig sperm
Authors: Mújica A. , Ruíz M. A. Valdes
Published: February 2005

In order to determine whether metabolizable sugars delayed capacitation of guinea pig spermatozoa, these cells were pre‐incubated in Tyrode's pyruvate lactate glucose medium (T‐PLG) or Tyrode's glucose solution (T‐G). They were then transferred to minimal culture medium containing pyruvate and lactate (MCM‐PL) and the occurrence of acrosomal reactions (AR) was determined by light microscopic observations of wet mount aliquots. The percentage of acrosomal reactions was quantitated in fixed samples and occurrence of a true AR was confirmed by electron microscopy. Activated acrosome‐reacted spermatozoa were observed within 5 min when cells were transferred to MCM‐PL solution, after preincubating them for 60–120 min either in T‐PLG or T‐G media. By 15 min in MCM‐PL the percentage of acrosome‐reacted spermatozoa reached values similar to those obtained in cells pre‐incubated from the beginning in MCM‐PL medium (P > 0.05 in both) but significantly different from T‐PLG and T‐G controls (P < 0.0005 in both). The acrosomal reaction was external calcium dependent and independent of the Tyrode's media pH ranging from 7.2 to 8.0. The results obtained suggested that capacitation occurred in T‐PLG and that it was not delayed by glucose; the results also suggested that capacitation could occur within a short time with glucose as the only exogenous substrate, but that the acrosome reaction could have been arrested by a glucose metabolite. Data are presented which suggest that intracellular levels of glucose‐6‐phosphate (as 2‐deoxyglucose‐6‐phosphate)could play a key role in the expression of the acrosome reaction in sperm already able to perform it. A new hypothesis is suggested for the development of the fertilizing potential of guinea pig sperm when in the female genital tract.
Evaluation of early embryonic mortality in donor insemination by a mathematical model, globally and depending on semen quality
Authors: Schwartz D. , Heuchel V. , Mayaux M. J. , O'Quigley J.
Published: February 2005

Stating the probability of pregnancy per cycle as PoPFPv, the product of the probabilities of ovulation, fertilization, and egg viability, the model allows an estimate of PF and PoPv for a series of cycles with known insemination timing. Such results obtained from a series of donor insemination (AID) compared with those generally admitted in natural reproduction suggest that the lower pregnancy rate in AID (all the lower when the postthaw motility is low) is owing to a lower egg viability. Since the abortion rate does not seem higher, there might be a sizable rate of very early embryonic deaths in AID perhaps even as early as nondeveloping eggs.
Species specificity of hybridoma antibodies to surface antigens of guinea pig sperm
Authors: Primakoff Paul , Heaton M. Rebecca , Myles Diana G.
Published: February 2005

The species specificity of hybridoma antibodies to sperm surface antigens was studied. A collection of over 50 hybridoma antibodies that bind to the guinea pig sperm surface was tested for binding to mouse, rat, hamster, and human sperm by indirect immunofluorescence. None of the antibodies bind to mouse sperm. rat sperm, or human sperm. All but three of the antibodies also fail to bind to hamster sperm. AH‐30, AH‐31, and AH‐1032, the three antibodies that crossreact with hamster sperm, show a different topographical localization on hamster sperm from that seen on guinea pig sperm. The three antibodies do not precipitate a 125I surface‐labeled antigen from hamster sperm extracts. However, from guinea pig sperm extracts, all three antibodies precipitate 125I surface‐labeled polypeptides with molecular weights (Mr) of 62,000, 52,000, and 38,000. This result suggests that the crossreacting antibodies may be recognizing different antigens on hamster and guinea pig sperm.
Spermiogenesis and the ultrastructure of mature sperm in Eurygaster intergriceps
Authors: Danilova L. V. , Gaber E. S. , Shinyaeva L. I.
Published: February 2005

An electron‐microscope study of spermiogenesis and the ultrastructure of mature sperm was made on Eurygaster integriceps. During spermiogenesis, a manchette consisting of two large groups of microtubules and an unusual centriolar adjunct are formed. The latter looks like two half cylinders located almost at right angles to one another. Its wall consists of several dark layers divided by lighter areas. The centriole and its adjunct are not identified in the mature sperm. Bug spermatids have a large amount of amorphous pericentriolar matter, which assists in establishing an unusual nuclear pattern.

The mature sperm is distinguished by a number of unique features. Its nucleus consists of three interconnected parts: the inner and outer cylinders and a part freely suspended along the middle piece. The intranuclear channel is blindly closed at the apical end and filled with dark amorphous matter that originates from the pericentriolar matter. The acrosome has an extracellular part resembling a diagonally striated rod, which is sometimes disengaged from its surface. The axoneme has 9+9(2) + 2 tubules. It is connected with the Nebenkern by dark arms.
Adenylate cyclase activity of membrane fractions isolated from sea urchin sperm
Authors: Mourelle Marisabel , Vargas Irma , Darszon Alberto
Published: February 2005

The adenylate cyclase activity of sperm membrane fragments isolated from Lytechinus pictus sperm according to Cross [20] has been studied. Two distinct fractions preferentially coming from the flagellar plasma membrane are obtained. Surface I125‐labeling experiments performed by Cross [20] indicate that these membranes are representative of the entire sperm plasma membrane. Both fractions are enriched in their adenylate cyclase activity: the specific activity of the top membranes is eightfold higher than in whole sperm, whereas that of the middle membranes is 15‐fold higher. The cyclase seems to be associated with the membranes. Lytechinus pictus egg jelly has no effect or slightly inhibits the adenylate cyclase activity of the isolated sperm plasma membrane fragments. Mg++ and Na+ stimulated their cyclase activity about sevenfold at 2.5 mM Mn++ and 3.2 mM ATP. At this ATP to Mn++ ratio, high concentrations of Ca++ have a small stimulatory effect.
Effects of cyclic AMP on the motility of mature and immature hamster epididymal spermatozoa studied by reactivation of the demembranated cells
Authors: Yeung C. H.
Published: February 2005

Hamster spermatozoa from the caput and cauda epididymides were demembranated with 0.04% Triton X‐100 and reactivated with 1 mM ATP. Motility parameters were analysed by video recording and stroboscopic photography. In the absence of added cAMP, reactivated cauda sperm showed percentage motility and forward swimming patterns similar to those of intact cells, but velocities were lower. When 2 or 20 μM cAMP was present, the velocities were increased but there was no effect on beat frequencies or percentage of forward progressing sperm. Cyclic AMP also markedly increased the percentage of cauda sperm which at first displayed nonprogressive “looping” movement. Addition of cAMP to the reactivation medium greatly improved the otherwise feeble and irregular motility of the demembranated caput sperm by increasing the percentage motility and beat frequencies of nonprogressive cells. It also induced forward motility with beat frequencies and velocities similar to cauda sperm reactivated in the absence of cAMP, but looping was never seen, indicating a change in the flagellar apparatus with maturation. The time required for the exhibition of the cAMP effects was reduced when caput sperm were reactivated in extracts of another previously maximally reactivated caput sperm preparation. The results suggest the production of some potent compound(s) by the axonemes for the manifestation of the cAMP effects.
Human sperm motility after migration into, and incubation in, synthetic media
Authors: Mortimer D. , Courtot A. M. , Giovangrandi Y. , Jeulin C. , David G.
Published: February 2005

Ten frames/sec microcinematography (“film”), 1 second timed‐exposure photomicrography (“photo”), and laser Doppler velocimetry (LDV) were used to analyse the swimming patterns of human spermatozoa after migration (1 h at 37°C) into an overlying layer of either BWW or Menezo's B2 media. The upper layer of medium was carefully removed and further incubated at 37°C for either 4 h (B2) or 5 h (BWW) and the sperm motility analysed again. Five experiments were performed using semen from different donors. Film and photo analyses gave the relative incidence of nonprogressive and progressively motile spermatozoa plus, for the progressive spermatozoa, the velocities of progression (Vp) and amplitudes of lateral head displacement (Ah). LDV gave the percentage of motile spermatozoa and the modal instantaneous velocity (Vm). All postmigration sperm populations showed large significant increases in the percentage of motile spermatozoa, with good survival during incubation. The progressive postmigration spermatozoa generally moved with greater Vp and Ah than in the initial seminal plasma‐diluted material; Vm was also increased. There were further increases in both Vp and Ah during incubation, but no change in Vm was detected. While the majority of spermatozoa were progressive, some showed a highly active pattern of movement which resulted in no net forward progression. The possible homology between these spermatozoa and the “hyperactivated” motility of capacitated spermatozoa in other mammalian species is discussed. Apparent discrepancies between the three methods used for motility analysis were seen, the possible causes and significances of which are also discussed.
Human nonovulatory oocyte‐cumulus complexes: Ultrastructure, macromolecular synthesis, and developmental potential
Authors: Tesařík Jan , Kopečny Václav , Dvořák Milan , Pilka Ladislav , Kurilo Ljubov F.
Published: February 2005

Human nonovulatory oocyte‐cumulus complexes were obtained from large antral follicles 34 h after the injection of hCG. Their ultrastructure, RNA and protein synthesis, and the ability of oocyte maturation and subsequent fertilization in vitro were the subject of this study. The oocyte nucleus was situated in the cell periphery, possessing one or two dense nucleoli. Intranucleolar vacuoles were sometimes present. Mitochondria and smooth endoplasmic reticulum were the most abundant cytoplasmic structures. The number of cortical granules was rather low. Cumulus cells showed responsiveness to exogeneous hCG/endogeneous LH stimulation as morphologic signs of the oocyte‐cumulus uncoupling and of the early luteinization. Furthermore, the oocytes were capable of meiotic maturation in vitro, and following subsequent in vitro insemination the majority of them were penetrated by spermatozoa. However, polyspermy was detected in most cases, and cleavage was not achieved. A developmental defect of nonovulatory oocytes was suggested by an extremely low level of RNA synthesis while continuing active protein synthesis by nonovulatory oocytes was demonstrated. In contrast, the associated cumulus cells synthesized both RNA and protein, suggesting that the majority of granulosa cells of the nonovulatory follicles was probably unaltered. It is hypothesized that the development of nonovulatory follicles after induction of ovulation might reveal the developmental pattern which would lead, in some cases, to ovulation of nonviable human oocytes both in stimulated and natural cycles.
Births after a prolonged delay between oocyte recovery and fertilization in vitro
Authors: Fishel S. B. , Edwards R. G. , Purdy J. M.
Published: February 2005

Two normal, healthy children have been born after an oocyte was collected from each of two patients during their natural menstrual cycle, incubated for many hours and then fertilized in vitro. Details of the follicular phase prior to oocyte recovery, fertilization and embryonic development in vitro, and replacement of the embryos are described in detail. One embryo cleaved rapidly after its delayed fertilization, with blastomeres of uneven size and cellular fragments. The luteal phase seemed to be weak in both patients.
Effect of follicle cells on the maturation and developmental competence of ovine oocytes matured outside the follicle
Authors: Staigmiller R. B. , Moor R. M.
Published: February 2005

The hypothesis that follicle cells play a central role in endowing the oocyte with developmental competence during maturation has been tested in coculture studies. Oocytes and follicle cells in various combinations were cultured for 24 h with gonadotrophins and estrogen in a nonstatic culture system. Developmental competence was assessed by transfer of oocytes to inseminated receipient ewes followed by embryonic examination 12 days later or at parturition.

Denuded and corona‐enclosed oocytes resume meiosis in culture but remain immature and developmentally incompetent. By contrast, oocytes supported by the cumulus and underlying granulosa (cumulus‐oocyte complexes) undergo full maturation and normal subsequent embryonic development (42.6% to embryos). Addition of supplementary follicle cells during culture (5 × 106 cells/ml medium) is without beneficial effect on denuded oocytes. However, supplementary cells confer competence on corona‐enclosed oocytes (37% to embryos).

A dual cellular requirement for full maturation involving both cell numbers and some direct cell‐oocyte contact is highlighted by our experiments. The results demonstrate further that the nonstatic system provides a simple but reliable method of producing large numbers of fully matured oocytes for both research and clinical purposes.