bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular associations in the rat oocyte‐cumulus cell complex: Morphology and ovulatory changes
Authors: Dekel Nava , Kraicer Peretz F. , Phillips David M. , Sanchez Ramon S. , Segal Sheldon J.
Published: February 2005

Cumulus‐oocyte complexes were isolated from the follicles of proestrous rats or from the oviducal ampullae of estrous rats. The zona pellucida of some complexes was dissolved before fixation. The follicular cumulus cells were seen to be held together mainly by long processes, which often extended over a distance of several cells. Large numbers of straight processes from the corona radiata cells, passing to the oocyte, surface, were seen in the space formerly occupied by the zona pellucida. Oocyte microvilli were uniformly short; none traversed the zona.

The postovulatory complexes were covered by amorphous extracellular material which also filled the spaces between the cells. By lysis of this material with hyaluronidase the cumulus cells were detached. The surfaces of these cells were covered with blebs.

By testing the ability of hyaluronidase to remove the corona cells from the zona pellucida of complexes isolated around the time of ovulation, it was found that the completion of retraction of the corona cells processes occurred in the oviduct, immediately after ovulation. It is suggested that the oviducal environment may influence the final step of the withdrawal of the corona cells' projections from the zona pellucida.
Oocyte‐surface factor responsible for 1‐methyladenine‐lnduced oocyte maturation in starfish
Authors: Morisawa Masaaki , Kanatani Haruo
Published: February 2005

Fully grown oocytes of the starfish Asterina pectinifera, undergo breakdown of their germinal vesicles and subsequent maturation on treatment with 1‐methyladenine (1‐MeAde). However, oocytes treated with seawater containing 0.010% Triton X‐100 lost the capacity to respond to 1‐MeAde and their germinal vesicles remained intact. These decapacitated oocytes once again ac‐quired the capacity to respond to l‐Me Ade when they were incubated in sea water containing the extract of fully grown oocytes treated with Triton X‐100, from which the Triton X‐100 was removed after extraction by means of Bio‐Beads SM‐2 (TXE). Recovery of the capacity was also observed after washing such TXE‐treated oocytes with sea water. These results suggest that some factor (probably 1‐MeAde receptor or its fragment), extracted from the oocyte surface (plasma mem‐brane) by nonionic detergent, was reconstituted on the oocyte surface so that the capacity of the oocytes to respond to 1‐MeAde was recovered. The factor was heat‐stable and resistant to treat‐ment with proteolytic enzymes.
Terminal deoxynucleotidyl transferase in rooster sperm
Authors: Witkin Steven S. , Reiner Brian , Brown Cynthia A.
Published: February 2005

A 165,000g pellet fraction, isolated from lysed rooster spermatozoa, was shown to contain a deoxynucleotide polymerizing activity which banded in sucrose at a buoyant density of 1.15gm/ml. The solubilized enzyme, partially purified by gel filtration, was estimated to have a molecular weight of 33,000 and utilized dT10 or dG10 as exogenous primers in template‐independent reactions sensitive to either sodium pyrophosphate or ethylenediaminetetra‐ cetic acid. Pyrimidine nucleotides (dTTP) were preferentially polymerized in the presence of Co++, while purine nucleotides (dGTP) were the preferred substrate when Mn++ was the divalent cation. These properties are all consistent with those of terminal deoxynucleotidyl transferase. An enzyme with similar properties was also obtained by subjecting a rooster sperm lysate to the procedure utilized to isolate terminal deoxynucleotidyl transferase from calf thymus. It is hypothesized that the enzyme may be involved in the regulation of gene expression during embryogenesis.
Sperm glycoproteins of the boar, bull, rabbit, and ram: I. Acrosomal glycoproteins
Authors: Fléchon Jacques‐Edmond
Published: February 2005

Ejaculated spermatozoa of boars, bulls, rabbits, and rams were fixed with glutaraldehyde and embedded in glycolmethacrylate. Thin sections were treated with phosphotungstic acid at low pH in order to localize cellular glycoproteins stained by the PAS technique at the light microscope level. At the ultrastructural level the glycoproteins were segregated in the anterior segment of mature spermatozoa. The distribution of these glycoproteins in the anterior segment was not homogeneous; it appeared species‐specific in detail, but as a rule, the maximum concentration was observed in a superficial layer, especially in the marginal thickening. The localization of other acrosomal components (eg, crystalline and basic proteins) is also reviewed. The origin and significance of the segregation of proteins and glycoproteins in the acrosome are discussed in relation to the fact that the acrosomal enzymes analyzed so far are glycoproteins.
The zymogen form of acrosin in testicular, epididymal, and ejaculated spermatozoa from ram
Authors: Harrison R. A. P. , Brown C. R.
Published: February 2005

The sperm‐specific proteinase acrosin (EC 3.4.21.10) is found in spermatozoa as a zymogen. We have looked for different forms of this zymogen in testicular, epididymal, and ejaculated spermatozoa from ram and have compared total sperm extracts made immediately after cell disruption with extracts made later from isolated sperm heads. We have concluded that the autoactivatable zymogen form, known generally as proacrosin, is the only form of acrosin within intact mature ram spermatozoa; no other zymogen form was detected, although lower levels of proacrosin were found in some samples of testicular spermatozoa. From studies of the activation process, it appears that ram proacrosin is truly autoactivatable; no evidence could be found for the involvement of any auxiliary enzyme. Estimations of the molecular weight of proacrosin using gel chromatography (60,000) and SDS‐polyacrylamide gel electrophoresis (51,300) indicated that the zymogen is monomeric. Comparison with the molecular weight of ram acrosin (44,000 or 40,000, using the two respective methods) indicated that a single acrosin molecule is derived from each zymogen molecule. The sperm acrosin inhibitor (molecular weight 11,000 or 8,000) was present in testicular spermatozoa as well as in ejaculated spermatozoa; there was no evidence that it was produced as a result of zymogen activation.
Multiple molecular forms of acid nitrophenyl phosphatase in sea urchin eggs and embryos
Authors: Yokota Yukio , Nakano Eizo
Published: February 2005

Acid nitrophenyl phosphatases from sea urchin eggs and embryos were investigated by gel filtration. Four different forms were found in Hemicentrotus pulcherrimus, and three forms in Anthocidaris crassispina and Pseudocentrotus depressus.

The first and second forms (designated AcP‐1 and AcP‐2) had the highest activity in the range of pH 5.6–6.0. The third (designated AcP‐3) had an apparent optimum pH between pH 4.3 and 4.8, and the fourth (designated AcP‐4) showed the maximum activity at pH 3.0. AcP‐1 was much more thermolabile than AcP‐2 and AcP‐3 at 56°C. NaF inhibited AcP‐2, AcP‐3, and AcP‐4 but not AcP‐1. AcP‐1, AcP‐2, and AcP‐3 were observed in the three species, whereas AcP‐4 was not detected in A. crassispina and P. depressus. AcP‐1, AcP‐2, and AcP‐3 were separted by gel filtration.

AcP‐1 and AcP‐2 of A. crassispina and H. pulcherrimus were studied in developing embryos. The behavior of these forms in gel filtration changed during development, from unfertilized eggs to the pluteus stage.
The spermatogenesis of crustaceans. VI. The external morphology of the mature spermatozoa of mytilicola intestinalis, steuer (copepoda)
Authors: López‐Camps J. , Bargallo R. , Fontarnau R.
Published: February 2005

The external structure of the male gamete of Mytilicola intestinalis is studied under a scanning electron microscope and compared with transmission electron micrographs of thin sections corresponding to the different parts of same. The cell in question is long and filiform, showing, along a significant part of its length, two ridges or expansions helicoidally arranged and diametrically opposed. Four different parts or segments can be identified in this spermatozoon: segment A, characterized by its screw‐like aspect; segment B, the longest, provided with well‐developed helicoidal expansions; segment C, showing an uneven surface and lacking expansions; and segment D, with a smooth surface and decreasing diameter. The significance of this original structure in a spermatozoon, considered immobile until now, is discussed, stating different hypotheses with regard to the possible mobility of the cell just before fertilization takes place.
Time and process of sperm penetration into cumulus‐free mouse eggs fertilized in vitro
Authors: Sato Kodo , Blandau Richard J.
Published: February 2005

Sperm penetration through the zona pellucida and fusion of the sperm head with the vitellus were observed continuously and filmed under phase optics in cumulus‐free living mouse eggs inseminated in vitro with capacitated epididymal sperm. Most spermatozoa penetrated the zona pellucida, traversed the perivitelline space, and fused with the vitellus at an angle nearly perpendicular to the surface. The mean duration required for sperm to penetrate the zona pellucida was 20 minutes with a range of 15–26 minutes. Sperm traversed the perivitelline space in less than one second. The initial contact of sperm with the vitellus generally took place at the tip of the sperm head. When the tip of the sperm head contacted the vitellus there was an immediate reduction in the rate of flagellation, followed by the gradual sinking of the sperm head into the vitellus.
The vitelline envelope, chorion, and micropyle of Fundulus heteroclitus eggs
Authors: Dumont James N. , Brummet Anna Ruth
Published: February 2005

The architecture and transformation of the vitelline envelope of the developing oocyte into the chorion of the mature egg of Fundulus heteroclitus have been examined by scanning and transmission electron microscopy. The mature vitelline envelope is structurally complex and consists of about nine strata. The envelope is penetrated by pore canals that contain microvilli arising from the oocyte and macrovilli from follicle cells. During the envelope's transformation into the chorion, the pore canals are lost and the envelope becomes more fibrous and compact and its stratified nature less apparent. The micropyle, of pore, through which the sperm gains access to the enclosed egg is located at the bottom of a small funnel‐shaped depression in the envelope. Internally, the micropyle opens on the apex of a cone‐like elevation of the chorion. During the development of the envelope, structured chorionic fibrils, the components of which are presumed to be synthesized by the follicle cells, become attached to its surface. These chorionic fibrils are though to aid in the attachment of the egg to the substratum and perhaps to help prevent water loss during low tides when the egg may be exposed.
Identification and hormonal control of reproductive‐tract‐specific antigens present in rabbit oviductal fluid
Authors: Stone Sarah Lipford , Huckle William R. , Oliphant Gene
Published: February 2005

Utilizing the intra‐abdominal flask technique to collect oviductal fluid, the presence of two or possibly three reproductive‐tract‐specific antigens have been observed in rabbit oviductal fluid. Two of these antigens may be accounted for by the two forms of uteroglobin. The other antigen has a molecular weight greater than 200,000 daltons and its concentration in oviductal fluid is under hormonal control. During pseudopregnancy (PSP), when progesterone concentrations are high, or upon progesterone administration, the concentration of this high molecular weight antigen doubles in oviductal fluid. This correlates well with the previously observed increase in release of secretory products from the oviductal epithelia.
A new procedure for rapidly scoring acrosome reactions of human sperm
Authors: Talbot Prudence , Chacon Richard
Published: February 2005

Because the acrosome of human sperm is too small to be directly visualized by phase‐contrast microscopy, acrosome reactions (that is loss of the acrosome) are generally not evaluated in studies of human sperm capacitation and fertilization. Nevertheless, it would be useful in such studies to have a technique for easily identifying and quantitating acrosome‐reacted sperm. In this paper, we describe a method for labeling the human sperm acrosome with fluorescein‐conjugated Ricinus communis agglutinin‐60 (FITC‐RCA); we show that in sperm without acrosomal caps, FITC‐RCA labeling occurs either not at all or only in the equatorial segment of the acrosome. To determine if the absence of FITC‐RCA labeling in the acrosomal cap region gives a reliable estimate of acrosome reactions, washed sperm or sperm incubated in a capacitating medium (BWW) were divided into two groups, which were then fixed for FITC‐RCA labeling or transmission electron microscopy. Counts of acrosome reactions made by each method were similar, and we observed an increase in the percentage of reactions following incubation in BWW. We conclude that the FITC‐TCA labeling technique is a reliable method for accurately scoring the percentage of acrosome‐reacted human sperm.
Gonadotropin stimulation of steroidogenesis and cellular dispersion in cultured porcine cumuli oophori
Authors: Hillensjö Torbjörn , Channing Cornelia P.
Published: February 2005

The relationship between cellular dispersion and steroidogenesis was studied in culture using oocyte‐cumulus complexes harvested from porcine follicles. The cells were cultured in modified TC 199 containing pig serum for one to two days. When oocyte‐cumulus complexes were cultured in the absence of hormone the oocytes resumed meiosis, the cumulus cells grew out forming monolayers, and progesterone accumulation was low. Addition of ovine LH, purified human LH, or purified human FSH stimulated expansion of the cumulus mass as well as enhanced progesterone accumulation. Oocyte maturation was not affected by the hormones. In absence of hormone, oocyte‐cumulus complexes obtained from large (6–12 mm) follicles showed increased cellular dispersion and higher progesterone accumulation as compared to complexes obtained from medium‐sized (3–5 mm) follicles.
Ultracytochemical localization of 3β‐hydroxy‐steroid ferricyanide reductase activity in the fetal mouse ovary
Authors: Hiura Masamichi , Jagiello Georgiana , Dennis James , Ducayen Mercedes
Published: February 2005

It has been suggested that the production of estrogens by the fetal ovary may modulate the entry or progression of meiosis in the female mammalian fetus. In the present study the possibility that the site of this steroid synthesis is the rete ovarii system was explored in the fetal mouse of gestational ages 12.5 to 18 days. The method of ultracytochemical localization of 3β hydroxy‐steroid ferricyanide was used. Reaction product was found in the cytoplasm of the rete ovarii (prefollicular) cells as early as day 14 with increasing amounts seen at later gestational ages. The presence of this essential enzyme system in cells closely applied to oogonia and oocytes during an active meiotic period must be considered in developing concepts of meiotic entry.
Aging of mouse eggs in vivo and in vitro
Authors: Longo Frank J.
Published: February 2005

Morphological and cytochemical (acid phosphatase) changes associated with mouse ova and cumulus cells aged within the oviducts (in vivo) or in culture (in vitro; 1–24 hours postovulation) have been investigated. Structural alterations of cumulus cells were apparent immediately after ovulation and included nuclear pycnosis and cytoplasmic vacuolization. Nevertheless, approximately 30% of the cumulus masses examined contained cells that plated out when cultured and remained viable for up t o three days in vitro. From 12 t o 24 hours postovulation almost all cumulus cells of specimens aged in vivo showed signs of degeneration. Disruption of the meiotic spindle and an increase in acid phosphatase positive organelles were characteristic of in vivo and in vitro aging ova. The percentage of fragmented eggs obtained from super‐ovulated (5 IU PMS followed by 5 IU HCG) mice approximately one and 24 hours postovulation was not significantly different. Eggs obtained from superovulated animals and aged in vitro for 24 hours yielded significantly more fragmented ova. Fragmented eggs were not obtained from cycling females on the morning of estrus. When such eggs were cultured in vitro for 24 hours the percent fragmentation was significantly lower than that for aged eggs obtained from super‐ovulated mice. These results indicate that 1) similar morphological alterations occur among cumulus cells and eggs aged either in vitro or in vivo, 2) ova from superovulated mice do not constitute a homogeneous population and 3) the method of superovulation employed in this study induces the ovulation of a relatively large group of eggs that are susceptible to fragmentation when cultured in vitro.
Preimplantation embryonic development of spontaneous mouse parthenotes after oocyte meiotic maturation in vitro
Authors: Eppig John J.
Published: February 2005

Of eggs ovulated in LT/Sv mice, 10–20% undergo spontaneous parthenogenetic activation, and 40–50% of the parthenotes develop to blastocysts when cultured in simple defined medium from the one‐cell stage. Similar percentages of oocytes isolated from Graafian follicles undergo parthenogenetic activation after spontaneous maturation in simple defined medium, but embryonic development proceeds no further than the two‐cell stage. The simple defined medium that supported preimplantation development of ovulated eggs and spontaneous maturation of extrafollicular oocytes contained no serum, free amino acids, or vitamins. The present experiments were conducted to determine what conditions during spontaneous maturation of extrafollicular oocytes could promote the ability of oocytes to develop to blastocysts after parthenogenetic activation and mimic the environment of preovulatory follicles.

Cumulus‐enclosed oocytes that were matured in simple medium supplemented with fetal bovine serum (FBS) developed to blastocysts after spontaneous parthenogenetic activation. Furthermore, minimum essential medium (MEM), a complex medium containing free amino acids and vitamins, could substitute completely for FBS for maturing oocytes from (C57BL/6J × LT/Sv)F1 mice, and to a lesser extent for maturing LT/Sv oocytes. Therefore, even though germinal vesicle breakdown in mouse oocytes and preimplantation development of mouse eggs can occur in the absence of an exogenous supply of free amino acids and vitamins, a complete, or normal, mouse oocyte maturation cannot. These results also demonstrated that gonadotropins are not necessary during oocyte meiotic maturation for parthenogenetically activated eggs to develop through the preimplantation stages.

Luteinizing hormone or 17β‐estradiol in MEM during oocyte maturation had no effect on the subsequent development of parthenotes. In contrast, follicle stimulating hormone (FSH) and progesterone in the maturation medium decreased the number of ova that subsequently cleaved, and FSH decreased the number of cleaved eggs that developed to blastocysts.
Sperm abnormalities in the PL/J mouse strain: A description and proposed mechanism for malformation
Authors: Burkhart J. G. , Malling H. V.
Published: February 2005

A high frequency (42%) of sperm from the inbred homozygous mouse strain PL/J are abnormal. Head shape abnormalities occur in 15% of the total sperm; and 27% of the sperm are headless, with the mitochondria condensed into a mass at the caudal end of the midpiece region. The sperm without heads exhibit relatively normal motility. Electron microscopy of the testes indicates that some of the abnormal sperm in PL/J males result from a failure of the paired centrioles to attain a normal position on the nucleus opposite the acrosome prior to implantation, or to attach at all. The centrioles that are not attached to the nuclear envelope can differentiate to form the principal piece and midpiece region. The frequency of headless variants in heterozygous F1 indicates that the trait is mainly recessive. The offspring from the backcross of the F1 to homozygous PL/J parents did not give a clear‐cut segregation pattern. The frequency of abnormal sperm in the F1 and the backcross is higher when the female parent is a PL/J.
The movement characteristics of rabbit spermatozoa before and after activation
Authors: Johnson L. L. , Katz D. F. , Overstreet J. W.
Published: February 2005

Spermatozoa were flushed with mineral oil from the lower isthmus of the rabbit oviduct at four hours postcoitus (pc) and 11 hours pc. Videotapes were made of sperm behavior in the native isthmic fluid and after dilution of the fluid with culture medium. The tapes showed that, initially, spermatozoa in the native isthmic fluid were virtually immotile, but immediately resumed movement on contact with the culture medium. Isthmic sperm motility then evolved over a five‐ to 10‐minute interval into the characteristic biphasic pattern of activated movement. Cine films of isthmic spermatozoa taken with a high‐speed camera were analyzed to determine flagellar beat frequency, maximum flagellar curvature, and swimming velocity. Progressiveness ratios and hydrodynamic power outputs were then calculated for individual spermatozoa. Two phases of activated sperm movement, a whiplash phase and a progressive phase, were identified and characterized. The power output of activated spermatozoa increased twentyfold in comparison with the preactivated state. The power output of activated spermatozoa did not differ between the two phases of activated movement.
Oocyte maturation inhibitor (OMI) activity in protein granulosa cells
Authors: Centola G. M. , Anderson L. D. , Channing C. P.
Published: February 2005

To determine the origin of oocyte maturation inhibitor (OMI), cumulus‐enclosed porcine oocytes from medium follicles were cultured for two days in Medium 199 alone, the low molecular weight (< 2,000 daltons) fraction of porcine follicular fluid (pFFL), or extracts of granulosa cells from small (1–2 mm), medium (3–5 mm), and large (6–12mm) antral follicles. Additionally, the cumulus‐oocyte complexes were grown in the presence of the low molecular weight fraction of “conditioned” medium from suspension cultures of medium follicle granulosa cells. The percent maturation in cultures with pFFL was significantly (P < .001) less than control cultures. Similarly, addition of the granulosa cell extracts at a 1/20 dilution resulted in a significant reduction in the percent oocyte maturation as compared with controls. The percent maturation after addition of conditioned medium was similarly reduced (P < .001). These results suggest that the granulosa cells probably synthesize and secrete OMI which inhibits oocyte maturation in vitro. Additionally, it appears that the content of OMI in the granulosa cells decreases as the follicle matures.
Acquisition of meiotic competence in the rat: Role of gonadotropin and estrogen
Authors: Bar‐Ami S. , Tsafriri A.
Published: February 2005

Oocytes were removed from the follicles of rats at 15 to 31 days of age, and their ability to resume meiosis (“meiotic competence”) in vitro was correlated with their diameter and the stage of follicular development. The majority of oocytes explanted on day 15 did not resume meiosis when placed in culture, but the percentage of competent oocytes increased from 14.1% ± 3.0% on day 20 to 67.6% ± 3.3% on day 26 of age. This ability to resume maturation correlated well (r = 0.98) with the increase in diameter of oocytes and coincided with the development of antral follicles.

Hypophysectomy on day 15 of age, but not on day 20, reduced the percentage (P < 0.001) and number (P < 0.001) of competent oocytes and was accompanied by a reduction in diameter of oocytes. Treatment with PMSG or E2 increased the number (P < 0.001) and percentage (P < 0.001) of competent oocytes. These results suggest that the ability of oocytes to mature in vitro is dependent upon stimulation by gonadotropins and that this action of gonadotropin may be mediated by production of estrogen within the follicles.
Effect of FSH and HCG on progesterone secretion by cultured oocyte‐cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development
Authors: Bae In‐Ha , Channing Cornelia P.
Published: February 2005

Oocyte‐cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2‐day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte‐cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3‐day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte‐cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness.

Monolayer formation ability of oocyte‐cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose‐dependent decrease in monolayer formation by oocyte‐cumulus complexes harvested from all sizes of follicles.
Glycosidase and cumulus dispersal activities of acrosomal extracts from opossum (marsupial) and rabbit (eutherian) spermatozoa
Authors: Rodger John C. , Young Ronald J.
Published: February 2005

Opossum and rabbit sperm sonicates dispersed the mouse cumulus oophorus in vitro at the same rate on a per sperm basis, despite much higher activities of the glycosidic acrosomal enzymes N‐acetylhexosaminidase (350 ×) and arylsulphatase (40 ×) in the opossum preparation. Activities of another glycosidase, hyaluronidase, and the protease acrosin where similar in sperm extracts from both species. However, specific inhibitors of N‐acetylhexosaminidase (iodoacetate) and arylsulphatase (SO42−, PO43−) markedly reduced the rate of cumulus dispersal by rabbit sperm sonicates. These findings suggest that, while hyaluronidase action probably represents the rate‐limiting (slow) step, several glycosidases act together to disperse the cumulus and in the passage of the fertilizing spermatozoon through it in eutherian mammals. The likely role of these acrosomal enzymes in the marsupial, where the ovulated oocytes lack a cumulus oophorus, is more uncertain.
Promotion of capacitation of guinea pig spermatozoa by the membrane mobility agent, A2C, and inhibition by the disulfide‐reducing agent, DTT
Authors: Fleming A. D. , Kosower N. S. , Yanagimachi R.
Published: February 2005

The membrane mobility agent A2C accelerates the onset of the acrosome reaction of guinea pig spermatozoa by promoting capacitation. Spermatozoa incubated in a suspension of A2C particles in Ca2+‐free medium for one hour undergo a synchronous, rapid acrosome reaction upon the addition of Ca2+. These acrosome‐reacted spermatozoa are capable of fertilization as assessed by their ability to penetrate (fuse with) zona‐free hamster eggs.

The disulfide‐reducing agent, dithiothreitol (DTT) inhibits A2C‐mediated capacitation. It also blocks fertilization of zone‐free eggs by acrosome‐reacted spermatozoa by preventing attachment of the spermatozoa to the egg plasma membrane.

The mode of A2C action on spermatozoa is compared to that of A2C‐induced fusion in somatic cells. The similarity of the molecular events in the sperm membrane during capacitation and the acrosome reaction to these in other fusion events is pointed out.

Inhibition of capacitation by DTT points to the importance of membrane and/or submembrane proteins and thiol groups in this process. Oxidation of sperm membrane SH groups may play an important role in in vivo capacitation.
Identification of surface autoantigens which appear during spermatogenesis
Authors: Romrell Lynn J. , O'Rand Michael G. , Sandow Pamela R. , Porter James P.
Published: February 2005

Autoantigens that appear during spermatogenesis in the rabbit were identified using immunoadsorbent chromatography and SDS‐PAGE. To identify cell‐surface proteins, samples of freshly isolated, staged cells were labeled by the lactoperoxidase or Iodo‐Gen iodination procedure and run on SDS‐PAGE. Autoradiograms of the stained, dried gels were prepared. By correlating the band patterns in the SDS gels of immunocolumn and surface‐labeled samples with the band patterns in the autoradiograms, it was possible to show when the autoantigenic proteins appeared on the cell surface. To further support the identification of membrane autoantigens, surface‐labeled, staged cell samples were lysed in Triton X‐100 and immunoprecipitated with antitestis cell autoantisera.

Three types of autoantigens have been identified: (1) late class antigens that are present only on late spermatids and epididymal spermatozoa, but are intracellular in early stages, (2) early class antigens which occur on the surface of pachytene spermatocytes and are present throughout subsequent stages of development, and (3) early class, transient antigens, which appear on spermatogenic cells but are not present on epididymal spermatozoa.
Zinc does not inhibit the capacitation of human spermatozoa in vitro
Authors: Blazak W. F. , Overstreet J. W.
Published: February 2005

The effects of zinc on human sperm motility, fertilizing capacity (as assessed by penetration of human spermatozoa into the zona pellucida‐free hamster oocyte), and nuclear chromatin decondensation were investigated using spermatozoa from four fertile donors. Both sperm motility and the penetration of sperm into zona‐free hamster ova were consistently impaired in media containing 1,000 μM zinc. Spermatozoa from one man were similarly affected at a concentration of 500 μM zinc, but no adverse effects were noted at this zinc concentration in experiments with other donors. Since decreased fertilizing capacity in response to zinc was always accompanied by a significant decline in both the percentage of motile cells and mean swimming speeds, it appears that all of these results reflect a general toxic effect on the cells. At lower concentrations (125–250 μM), zinc had no effect on human sperm motility nor their ability to undergo capacitation and penetrate zona‐free hamster ova in vitro. For some donors, zinc (125–500 μM) stimulated both the attachment of spermatozoa to the hamster vitellus and the incorporation of spermatozoa into the hamster ooplasm. The decondensation of human sperm nuclear chromatin in sodium dodecyl sulfate was largely inhibited when zinc was added to the medium, but no significant changes in nuclear stability were apparent after capacitation in zinc‐free medium. We conclude that zinc, when present in subtoxic concentrations, does not adversely affect the ability of human spermatozoa to undergo capacitation and penetrate zona‐free hamster ova in vitro.
Ultrastructure of follicular atresia in the rat
Authors: Gondos Bernard
Published: February 2005

Observations were made on the sequence of morphologic changes in atresia of medium‐sized preantral follicles in the rat. Ultrastructural studies indicated that in both control and hormonally treated animals granulosa cell changes, including nuclear condensation and alterations in cytoplasmic organelles, occurred prior to effects on the oocyte. In more advanced stages of atresia, extensive disruption of granulosa cell cytoplasm was associated with loss of microvilli and cytoplasmic vacuolization in oocytes. The findings are consistent with the view that follicular atresia begins with alterations in granulosa cells, effects on the oocyte occurring later in the atretic process.
Influence of luteinizing hormone and progesterone on maturation and fertilizability of rat oocytes in vitro
Authors: Barak Yona , Kaplan Ruth , Kraicer P. F. , Shalgi Ruth
Published: February 2005

The effects of luteinizing hormone (NIH‐bovine LH) and progesterone on maturation in vitro of oocyte‐cumulus complexes from adult proestrous rats were studied by comparing proportions of oocytes showing germinal vesicle breakdown, mucification of the cumulus oophorus, and fertilizability. Addition of either or both of the hormones to the medium in concentrations between 1.25 and 10 μg/ml during maturation had no discernible effect on germinal vesicle breakdown or on fertilization. Mucification was stimulated by LH and even more by LH plus progesterone. It was concluded that maturation in vivo is the result of concerted action of the two hormones. However, addition of LH + progesterone had no effect on the fertilizability of these oocytes. We attribute this to a relative insensitivity of the system for fertilization in vitro to subtle changes in the oocyte.
Stage‐specific changes in protein phosphorylation during preimplantation development in the mouse
Authors: Lopo Alina C. , Calarco Patricia G.
Published: February 2005

To gain insight into the role of protein phosphorylation during early mammalian development, seven mouse preimplantation stages were metabolically labeled with radioactive orthophosphate and the radiolabeled proteins identified using gel electrophoresis and autoradiography. The results obtained indicate that there are marked differences in protein phosphorylation patterns between the zygote and two‐cell stage and between the morula and blastocyst stage. In addition, there is a compaction‐specific change in the phosphorylation profile of three components of Mr 37,000. This compaction‐specific change takes place during compaction in the eight‐cell embryo; thus, it is the first biochemical change specifically correlated to this important event of early development.
Ultrastructure of the head of ctenomys maulinus spermatozoon with special reference to the nucleus
Authors: Feito R. , Barros Claudio
Published: February 2005

The spermatozoa of the neotropical hystricomorph rodent Ctenomys maulinus have been examined cytochemically and under the transmission electron microscope. The head is flattened dorsoventrally. At the caudal end of the head there is a process oriented parallel to the tail. This process corresponds to a cylindrical extension of the nucleus, which constitutes a unique feature among mammals.
Microdensitometric evaluation of the DNA content, as ploidy parameter, of spermatozoa in the polymorphic chromosomal system of Akodon molinae cabrera (rodentia, cricetidae)
Authors: Redi C. A. , Garagna S. , Merani M. S. , Capanna E. , Bianchi N. O. , Romanini M. G. Manfredi
Published: February 2005

The influence of chromosome variability on the production of euploid spermatozoa was investigated in a suitable biological model, the Akodon molinae system. This consists of individuals whose chromosome constitution is 2n = 42, 2n = 43, or 2n = 44. The only difference between these three karyotypes occurs through a Robertsonian rearrangement combined with two pericentric inversions. Thus, the animals with 2n = 42 (simple homozygotes or SH) have two large metacentric chromosomes number 1; animals with 2n = 43 (heterozygotes or Ht) have a chromosome 1 and two subterminal chromosomes la and lb homologues of the long and short arms of the chromosome 1, respectively; animals with 2n = 44 (double homozygotes or DH) have a pair of la and a pair of lb chromosomes. The gametic euploidy frequency correlated with each chromosome constitution was evaluated on the basis of the DNA content of spermatozoa, which was determined microdensitometrically after the Feulgen reaction, taking into account the site of the spermatozoa along the male genital tract. A comparative assessment of gametic aneuploidy frequency in caput epididymis versus vas deferens demonstrated (1) a falloff in euploid production in passing from the 2n = 42 to the 2n = 44 chromosome forms, alongside a high degree of intragroup variability, and (2) a lower aneuploidy frequency in the vas deferens than in caput epididymis in all the forms considered. These two features, taken together with similar results in the mouse chromosome variability system, suggest that a selection mechanism is operative against aneuploid spermatozoa in the epididymis. This finding is of interest in a wider perspective, since it might turn out to be valid for many mammals.
Timing of human sperm chromosome replication following fertilization of hamster eggs in vitro
Authors: Balkan Wayne , Martin Renée H.
Published: February 2005

The visualization of human male pronuclear chromosomes is possible by utilizing a technique in which human sperm fertilize zona‐free Golden hamster (Mesocricetus auratus) ova in vitro. R banding of these chromosomes can be achieved by adding 20 μg/ml 5‐bromode‐oxyuridine (BrdU), a thymidine analogue, to the culture medium during mid‐to‐late S phase and subsequently staining the chromosomes with 0.1 mg/ml acridine orange. BrdU was added 3.0–10.0 hr postinsemination (hr pi). R bands were obtained when fertilized eggs were transferred into medium containing BrdU between 5.0 and 6.5 hr pi. This indicates that the human (male) pronuclear chromosomes were in mid‐to‐late S phase at these times.
Evaluation of premeiotic activity in the developing rabbit testis using a modified squash technique
Authors: Randolph Margaret , Gondos Bernard
Published: February 2005

Germ cells in the developing rabbit testis were studied using a modified squash technique. Preleptotene figures present in the postnatal testis were examined and compared with corresponding stages in ovarian germ cells. The findings indicated differences in the pattern of preleptotene changes in the developing ovary and testis.
22Na+and86Rb+fluxes in spermatozoa and oocytes of arbacia punctulata
Authors: Adeyemo Oyewole , Koide S. S.
Published: February 2005

The fluxes of22Na+and86Rb+in Arbacia sperm and oocytes were studied in order to determine how these cells carry out cation exchange with the sea environment. The uptake of these ions by serum followed a pattern of early rapid influx (initial 0.5 min) and subsequent efflux (1–3 min) followed by a gradual uptake (after 3 min). Neither the uptake nor the efflux of these cations by Arbacia sperm were affected by ouabain, suggesting that influx and efflux of22Na+and86Rb+in Arbacia sperm occur predominantly by passive transport. The22Na+uptake by Arbacia oocytes showed a steady increase after an initial rapid uptake. A slight but significant inhibition of22Na+uptake was observed with ouabain. However,86Rb+uptake by the oocytes reached an early equilibrium and was not affected by ouabain. The uptake of Rb+by Arbacia oocyte is by passive transport while that of Na+is both by passive and active transport.
Fusion of hamster and pig zona‐free eggs stimulated by boar and guinea pig sperm at fertilization in vitro
Authors: Pavlok Antonin , Trávník Pavel , Kopečnčy Václav , Šťastná Jitka
Published: February 2005

In the course of in vitro fertilization of zona‐free hamster and pig eggs by boar and guinea‐pig spermatozoa it was observed that homologous and heterologous eggs fused together, forming cell hybrids between two or more cells. The fusogenic activity was attributed to spermatozoa and this was the hypothesis tested. The fusogenic activity (coinciding with sperm penetration activity) was dependent on the duration of sperm preincubation, which may be regarded as capacitation in vitro. Fusion occurred only after 3 hr of sperm preincubation and a narrow optimum was detected at 4–4.5 hr. Fusion of eggs was also dependent on sperm concentration. A relatively high proportion of fusions was observed at a sperm concentration of 4.0 × 104 per ml and an optimum was attained at a concentration of 5.0 × 105 per ml. The first fusions were observed at 90 min after semination. After 3 hr more than a half of the eggs reacted, and by 20 hr of incubation 80% of ova were fused. The fusability of eggs was tested and found to occur at 14 hr after ovulation. The fusion process was also studied using transmission electron microscopy. It is supposed that the process of egg fusion may be caused either by a similar mechanism to sperm‐egg fusion, or by products released during the sperm acrosome reaction.
Isolation of motile spermatozoa by density gradient centrifugation in Percoll®
Authors: Lessley Bruce A. , Garner Duane L.
Published: February 2005

A procedure using centrifugation in density gradients composed of Percoll was developed for isolation of spermatozoa from mammalian semen. To evaluate the technique, rabbit, human, or bovine semen was layered over continuous Percoll gradients ranging in density from 1.02 to 1.13 gm/ml and centrifuged at 1,500g for 45 min. After centrifugation, the seminal plasma remained above the gradient, whereas the spermatozoa and seminal particles were distributed within the gradient according to their buoyant densities. Unlike most washing techniques, no sperm pellet was formed; instead, the spermatozoa were concentrated into a compact band above the most dense layer of Percoll. The spermatozoa recovered from the gradient were easily resuspended by gentle techniques. Thus, the mechanical stress to the spermatozoa was minimized. Osmotic stress to the spermatozoa was also negligible as the Percoll gradients were isotonic throughout. Spermatozoa obtained by this technique possessed motility equivalent to that of spermatozoa in the unfractionated semen. Sperm suspensions recovered from the gradients contained less than 5% of the nonspermatozoal particles present in the original samples of unfractionated semen. Soluble seminal components were also efficiently removed from the spermatozoa. Thirty billion bovine spermatozoa could be fractionated on a single gradient without loss of effectiveness. Recovery of spermatozoa from these preparative separations averaged 80%. These results demonstrated that Percoll was a superior medium for efficient density gradient isolation of motile spermatozoa free of contamination by other seminal constituents.
Adenylate cyclase activity in porcine sperm in response to female reproductive tract secretions
Authors: Berger T. , Clegg E. D.
Published: February 2005

Adenylate cyclase activities were studied in porcine sperm in the presence and absence of Mn++ before and after incubation in vivo and in vitro. Incubation of sperm in vivo for 30 min increased the Mg++‐stimulated adenylate cyclase activity from 35.1 pmoles cyclic AMP formed per mg protein per 10 min to 50.4 pmoles. The activity stimulated by Mg++ and Mn++ increased from 392 to 729 pmoles after 30 min of in vivo incubation. Activity after incubation in vivo for 120 min was not different from activity after 30 min. In vitro incubation of porcine sperm in Ca++‐free Ringer‐fructose resulted in no change, but incubation in oviductal and uterine flushings obtained from gilts soon after ovulation increased Mg++‐stimulated activity by 24% and Mg++−+ Mn++‐stimulated activity by 49%. In vitro incubations in preovulatory flushings plus follicular fluid or in bovine serum albumin also increased adenylate cyclase activity.
Effect of Na+‐free seawater on energy metabolism in sea urchin spermatozoa with special reference to coenzyme A and carnitine derivatives
Authors: Mita Masatoshi , Yasumasu Ikuo
Published: February 2005

When the dry sperm of sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial seawater, they became motile and the level of ATP decreased. However, after dilution in Na+‐free seawater, the sperm were still immotile and the ATP level remained constant. CoA, carnitine, and their derivatives as intermediates of lipid metabolism were also measured in sperm. The levels of CoA derivatives were much higher than those of carnitine derivatives. The free and acid‐soluble CoA level decreased and the long‐chain acyl‐CoA level increased after dilution in artificial seawater, However, they did not change after dilution in Na+‐free seawater. The levels of carnitine derivatives hardly changed after the dilution in both sorts of artificial seawater, The respiratory rate was extremely low in Na+‐free seawater, Furthermore, concentrations of pyruvate, lactate, citrate, and malate remained essentially constant after dilution in the Na+‐free seawater, whereas they changed markedly after dilution in the artificial seawater.
Nucleoprotein changes during male pronuclear development as determined by the ammoniacal silver reaction
Authors: Longo Frank J.
Published: February 2005

Sperm nucleoprotein changes during male pronuclear development in fertilized sea urchin (Arbacia punctulata) eggs have been examined utilizing the ammoniacal silver reaction (ASR) at the light and electronic microscopic levels of observation. Previous studies and control preparations indicated that the ASR has an affinity for basic proteins, staining intensely those rich in arginine residues. Differences in the affinity of the paternally derived chromatin to the ASR prior to, during, and following pronuclear development were observed. Relative to the female pronucleus the unincorporated sperm nucleus was densely stained. Upon its entry into the egg the sperm nucleus showed a two‐fold increase in staining, indicating an augmentation in the availability of reactive sites already present in the paternally derived chromatin or an accumulation of “new” reactive sites from the egg cytoplasm. With the dispersion of the sperm nucleus there was a progressive decrease in staining intensity of the paternally derived chromatin. Subsequent to pronuclear fusion the paternally derived chromatin, recognized by its relatively dense staining, was seen at one pole of the zygote nucleus. With time there was a gradual regression in the size and staining intensity of the paternally derived chromatin within the zygote nucleus. Changes in reactivity of sperm‐derived chromatin are discussed in reference to previous studies of chromatin transitions at fertilization.
Lysophosphatidylserine inhibits completion of meiotic maturation of rat oocytes in vitro
Authors: Fleming Alan D. , Armstrong David T.
Published: February 2005

Follicular oocytes collected prior to the expected time of the LH surge from PMSG‐treated immature rats were incubated cummulus‐intact (with or without LH) or cumulus‐free (CF). Oocytes were incubated in the presence or absence of lysophosphatidlylserine (LS), a naturally occurring membrane phospholipid that has been previously shown to block sperm‐related membrane fusion events. Fusion events occurring during oocyte maturation that might be affected by LS include maintenance of the intact germinal vesicle (GVI) and prevention of GV breakdown (GVBD) and first polar body formation (PBI). LS had only a slight effect upon GVI. The incidence of GVI was significantly increased in only one of the three oocyte culture conditions employed (CF). Exposure to LS from the outset of collection and washing did not increase the incidence of GVI, indicating the lack of effect by LS was not owing to the passage of a sensitive period during oocyte collection. In contrast, LS was not owing to the passage of a sensitive period during oocyte colection. In contrast, LS almost completely abolished PBI in all oocyte culture conditions at 100 μ in PBI and those sperm‐related fusion processes previously found to be sensitive to LS. Finally, LS or similar agents may be responsible for the block to maturation (often at anaphase I) and even the retarded cleavage observed in vitro during oocyte maturation or embryo culture in some species.