bluuebunny/crossref_metadata_2025 huggingface dataset:
A new embryonic antigen, p67, present during mouse preimplantation development
Authors: Banka Carole L. , Calarco‐Gillam Patricia D.
Published: February 2005

An antibody prepared against nullipotential teratocarcinoma stem cells (A‐N1) detects cell surface antigens expressed by early mouse embryos and inhibits in vitro development of embryos in the absence of complement [Calarco and Banka, 1979]. Here we report the immunoprecipitation and electrophoretic characterization of A‐N1‐detected antigens from preimplantation mouse embryos. Predominant antibody activity is directed against a 67,000‐dalton glycoprotein (p67) with a mean pI of 5.3, which has not been previously described. This protein is not detected, at least as p67, after culture of embryos in tunicamycin. The p67 antigen is also expressed by pluripotential PSA1 teratocarcinoma cells but not by several different differentiated mouse cell types.
A comparison of fertility in vitro and in vivo after exposure of male mice to high environmental pressure
Authors: Fraser Lynn R. , Monk Susan , Wardley‐Smith Bridget , Cohen Shara , Halsey Michael J.
Published: February 2005

Male mice of the TO strain were exposed to 50 ATA helium (0.5 ATA oxygen) over 5 weeks and then assessed for fertility both in vitro and in vivo. Significantly lower values for acrosome loss and fertilization of zona intact eggs in vitro were obtained with spermatozoa from the pressure‐treated males. These results, coupled with the significant decrease in incidence and degree of polyspermy in zona‐free eggs, indicated that fewer competent cells were present after preincubation under capacitating conditions. Fewer motile cells were observed in most samples, along with a reduction in the incidence of hyperactivated motility in some. Testis weight was also significantly lower. Results of fertilization in vivo, after mating with untreated females, showed a consistent trend toward subfertility, with a lower pregnancy rate and a smaller litter size. These differences did not reach significance, however, unlike those obtained in earlier in vivo experiments with males of the BALB/c strain. The discrepancy between in vitro and in vivo results with TO males probably represents differences between unselected and selected sperm populations, while that observed in vivo between the two strains probably reflects the superior reproductive capacity of the TO strain.
Adenosine and gpp(NH)p modulate mouse sperm adenylate cyclase
Authors: Stein D. M. , Fraser L. R. , Monks N. J.
Published: February 2005

The possible roles of adenosine and the GTP analogue Gpp(NH)p in regulating mouse sperm adenylate cyclase activity were investigated during incubation in vitro under conditions in which after 30 min the spermatozoa are essentially uncapacitated and poorly fertile, whereas after 120 min they are capacitated and highly fertile. Adenylate cyclase activity, assayed in the presence of 1 mM ATP and 2 mM Mn2+, was determined by monitoring cAMP production. When adenosine deaminase (1 U/ml) was included in the assay to deplete endogenous adenosine, enzyme activity was decreased in the 30‐min suspensions but increased in the 120‐min samples (P < 0.02). This suggests that endogenous adenosine has a stimulatory effect on adenylate cyclase in uncapacitated spermatozoa but is inhibitory in capacitated cells. Since the expression of adenosine effects at low nucleoside concentrations usually requires guanine nucleotides, the effect of adding adenosine in the presence of 5 x 10–5 M Gpp(NH)p was examined. While either endogenous adenosine or adenosine deaminase may have masked low concentration (10−9−10−7 M) effects of exogenous adenosine, a marked inhibition (P < 0.001) of adenylate cyclase activity in both uncapacitated and capacitated suspensions was observed with higher concentrations (>10−5 M) of adenosine. Similar inhibition was also observed in the absence of Gpp(NH)p, suggesting the presence of an inhibitory P site on the enzyme. In further experiments, the effects of Gpp(NH)p in the presence and absence of adenosine deaminase were examined. Activity in 30‐min suspensions was stimulated by the guanine nucleotide and in the presence of adenosine deaminase this stimulation was marked, reversing the inhibition seen with adenosine deaminase alone. In capacitated suspensions the opposite profile was observed, with Gpp(NH)p plus adenosine deaminase being inhibitory; again, this was a reversal of the effects obtained in the presence of adenosine deaminase alone, which had stimulated enzyme activity. These results suggest the existence of a stimulatory adenosine receptor site (Ra) on mouse sperm adenylate cyclase that is expressed in uncapacitated spermatozoa and an inhibitory receptor site (Ri) that is expressed in capacitated cells, with guanine nucleotides modifying the final response to adenosine. It is concluded that adenosine and guanine nucleotides may regulate mouse sperm adenylate cyclase activity during capacitation.
Nuclear morphogenesis during spermiogenesis in the nudibranch molluscHypselodoris tricolor (Gastropoda, opisthobranchia)
Authors: Medina A. , Moreno J. , López‐Campos J. Ló
Published: February 2005

An electron microscope study was carried out on Hypselodoris tricolor spermatids to describe the development of the nuclear morphogenesis and investigate the possible cause(s) of the change in the shape of the spermatid nucleus during spermiogenesis. Three different stages may be distinguished in the course of the nuclear morphogenesis on the basis of the morphology and inner organization of the nucleus. Stage 1 spermatid nuclei are spherical or ovoid in shape and the nucleoplasm finely granular in appearance. Stage 2 nuclei exhibit a disc‐ or cup‐shaped morphology, and the chromatin forms short, thin filaments. During stage 3, a progressive nuclear elongation takes place, accompanied by chromatin rearrangement, first into fibers and then into lamellae, both formations helically oriented. A row of microtubules attached to the nuclear envelope completely surrounds the nucleus. Interestingly, the microtubules always lie parallel to the chromatin fibers adjacent to them. Late stage 3 spermatids show the highest degree of chromatin condensation and lack the manchette at the end of spermiogenesis. Our findings indicate the existence of a clear influence exerted on the chromatin by the manchette microtubules, which appear to be involved in determining the specific pattern of chromatin condensation in Hypselodoris tricolor.
19‐Norandrostenedione (4‐estrene‐3,17‐dione) Inhibits Porcine Oocyte Maturation In Vitro
Authors: Daniel S. A. J. , Khalil M. W. , Armstrong D. T.
Published: February 2005

Recent work has shown that 19‐norandrostenedione is a major steroidal component of porcine follicular fluid; however, little is known about its role(s) in the regulation of follicular function. This study was designed to examine the effect of 19‐norandrostenedione on porcine oocyte maturation in vitro. Oocyte‐cumulus complexes were isolated from medium (3–6‐mm diameter)‐sized prepubertal pig follicles and incubated for 12 h in medium with or without dibutyryl cyclic AMP ((Bu)2cAMP, 1 mM) with or without testosterone (5 x 10−7 M) or 19‐norandrostenedione (5 x 10−7 M). In medium alone, 70.8% of oocytes spontaneously resumed meiosis as evidenced by the occurrence of germinal vesicle breakdown. Oocyte maturation was inhibited by (Bu)2cAMP (44.6% of oocytes matured). Although neither steroid alone affected maturation, both testosterone and 19‐norandrostenedione enhanced the effect of (Bu)2cAMP (22.5 and 19.6%, respectively, resumed meiosis).

The effects of testosterone and 19‐norandrostenedione on (Bu)2cAMP‐inhibited oocyte maturation were dose dependent and there was no significant difference between the actions of the steroids. The effect of 19‐norandrostenedione was reversible and dependent on the presence of an intact cumulus. Hydroxyflutamide (SCH‐16423), a nonsteroidal compound known to block androgen receptors, abolished the effects of both testosterone and 19‐norandrostenedione on germinal vesicle breakdown, indicating that the actions of these steroids are truly androgenic.

The results of this study suggest that 19‐norandrostenedione may be of physiological importance in the regulation of porcine oocyte maturation.
Immunochemical identification of multiple cell surface antigens appearing during specific stages of mouse spermatogenesis
Authors: O'Brien Deborah A. , Millette Clarke F.
Published: February 2005

Cell surface antigens that appear in a defined temporal sequence during mouse spermatogenesis were previously detected serologically, but not identified biochemically, with four heterologous antibodies prepared against purified populations of pachytene spermatocytes (AP), round spermatids (ARS), vas deferens spermatozoa (AVDS), and mixed seminiferous cells (ASC) [Millette and Bellvé, J Cell Biol 74:86–97, 1977]. These antigens have now been identified immunochemically on nitrocellulose blots from SDS polyacrylamide gels. Three antisera (AP, ARS, and ASC) recognize a similar subset of determinants on one‐dimensional immunoblots of germ cells and plasma membranes prepared from a mixed population of late spermatogenic cells. Comparisons of minor bands to reveal differences among these antisera. AVDS exhibits the least complex binding pattern. The results indicate that at least ten surface constituents appear during the pachytene stage of meiosis, coincident with a period of maximal RNA and protein synthesis [Monesi, Exp Cell Res 39:197–224, 1965]. Furthermore, two‐dimensional immunoblot comparisons of plasma membranes isolated from pachytene spermatocytes and round spermatids reveal differences between surface determinants detectable at these two spermatogenic stages. For example, ASC recognizes two newly described proteins that are restricted to pachytene spermatocytes (˜ Mr 57,000, pI 6.45) and to round spermatids (˜ Mr 39,500, pI 4.85), respectively.
Effects of carbon dioxide acidification of mouse oocytes before in vitro fertilization, culture, and transfer
Authors: Puissant F. , Degueldre M. , Buisson L. , Leroy F.
Published: February 2005

The effects of varying pH levels on superovulated mouse oocytes before in vitro fertilization, culture, and transfer were investigated. Mouse oocytes acidified for 1 hour with 20% CO2 (pH 6.9) exhibited a wide range of alterations. Five hours after insemination, 25% of these eggs showed different degrees of cytolysis and their perivitelline space contained numerous spermatozoa. At 12 hours normal‐looking eggs from this group showed a higher proportion of polypronuclear zygotes than the control group (exposed, 51%; controls, 18%). Acidification thus markedly increased the permeability of the zona pellucida and/or interfered with the normal block to polyspermy. Fewer eggs achieved the two‐cell stage among groups submitted to acidotic pH levels (6.9 to 6.6). In contrast, alkaline conditions (pH 7.8) did not reduce the proportion of two‐cell embryos. However, only eggs maintained at pH 7.5 were capable of producing 80% of blastocysts. In other groups developmental blockage occurred mainly between stages 2 and 4. A significant reduction was found in the proportion of recipient females becoming pregnant after intrauterine transfer of blastocysts originating from CO2‐treated oocytes. This difference might be related to the existence of a large polyploid population among experimental eggs.
Variables affecting the yield and developmental potential of embryos following superstimulation and in vitro fertilization in rhesus monkeys
Authors: Boatman Dorothy E. , Morgan Patricia M. , Bavister Barry D.
Published: February 2005

Three follicular‐stimulation protocols were compared to evaluate the yield and quality of oocytes obtained from rhesus monkeys. Five animals received a high‐dose regimen of PMSG (protocol I), three received a lower‐dose regimen (protocol II), and two received Clomid‐Pergonal (protocol III). Oocytes were recovered at laparoscopy after HCG injection, fertilized in vitro, and cultured up to the blastocyst stage. Yields of mature oocytes were 17.2 ± 13.0 (80% of total recovered), 6.7 ± 6.6 (41%), and 4.5 ± 2.5 (90%) per stimulated cycle for protocols I, II, and III, respectively. Of mature oocytes, 72%, 45%, and 89% were fertilized for protocols I, II and III, respectively. Protocol I produced the most fertilized oocytes per stimulated cycle (11.6 ± 11.6) and the greatest E2 production (approximately fourfold that maximally expected for an unstimulated cycle). For the combined protocol I and II results, there was a significant correlation (P ⩽ 0.05) between mean embryo development score and E2 production. Fertilized oocytes from protocol I yielded 7.8 ± 8.0 morulae and 6.8 ± 7.2 early zonal blastocysts per cycle. After transfer of nine singleton embryos to surrogate recipients, one live birth resulted. We conclude that our high‐dose PMSG regimen offers the best means at present for obtaining susbstantial numbers of developmentally competent oocytes in rhesus monkeys and that more extensive use of rapid serum E2 assays for monitoring both stimulated cycles and those of potential surrogate recipients could help to predict the success of in vitro fertilization and embryonic development following embryo transfer in rhesus monkeys.
Interaction of boar spermatozoa with porcine oocytes: Increase in proteins with high affinity for the zona pellucida during epididymal transit
Authors: Peterson R. N. , Hunt W. P. , Henry L. H.
Published: February 2005

Cauda boar sperm but not caput sperm bind to isolated porcine oocytes, and several cauda sperm plasma membrane proteins (PMPs) with high affinity for isolated zonae have been identified. These PMPs migrate with apparent molecular weights near 70, 45 and 30 K [Peterson et al, Gamete Res 12: 91–100, 1985]. When caput plasma membranes (PM) were fractionated on dextran sulfate (DS) agarose and compared to cauda PM similarly fractionated, less protein with high affinity for DS bound to the column, and significant differences in the PMPs eluted with increasing concentration of NaCl were observed. The PMPs at ∼70, ∼45, and ∼30 K, noted above, and two bands near ∼20K were present in fractions from cauda PM but were absent or present in only small quantities in caput PM. Cauda PM fraction eluted at high salt were effective in blocking the binding of sperm to isolated oocytes; caput PM fractions were not. Antibodies to the cauda PMPs eluted at high salt, which blocks the binding of capacitated sperm to eggs and fertilization in vitro, were absorbed by cauda PMV but not by caput PMV. These findings suggest that the ability of boar sperm to attach to porcine oocytes develops as the result of the addition of one or more of these PMPs to sperm during epididymal transit.
Changes in elemental composition of fertilized and unfertilized northern pike (Esox lucius) eggs incubated in buffer with and without dimethyl sulfoxide: An X‐ray microanalysis study
Authors: Schmehl M. K. , Graham E. F.
Published: February 2005

Fertilized and unfertilized eggs from the northern pike (Esox lucius) were incubated 2 hr in buffer with 0 and 10% (v/v) dimethyl sulfoxide and then quickly frozen in the wells of aluminum blocks submerged in liquid nitrogen. Control eggs and ovarian fluid were similarly frozen immediately after collection. The frozen eggs were sectioned, freeze dried, mounted on stubs, and carbon coated. X‐ray microanalysis was used to determine changes in element levels and dimethyl sulfoxide (Me2SO) penetration in the zona radiata, cytoplasm, cortical alveoli, and egg yolk. Unfertilized eggs incubated without Me2SO showed decreased levels of Na, Cl, and K in the zona radiata; fertilized eggs, incubated without Me2SO showed decreased levels of Na, P, and Cl in the zona radiata and increased levels of K in the cytoplasm; unfertilized eggs, incubated with 10% Me2SO showed decreased Na and Cl in the zona radiata, decreased K in the cytoplasm and increased K in the cortical alveoli; fertilized eggs incubated in buffer with 10% Me2SO showed decreased levels of Na, P, Cl, and K (zona radiata), P, Cl, and K (cytoplasm), Na (yolk), and increased Cl in the yolk (all P<.01). Me2SO (v/v) levels reached 1.5‐3.1% in the zona radiata, 0‐3.2% in cytoplasm, 2.3‐8.7% in cortical alveoli, and 0‐1.6% in the yolk. Unfertilized eggs showed more Me2SO penetration than fertilized eggs.
Hamster oocyte fertilizability after 4°C storage
Authors: Barros C. , Herrera E. , Fuenzalida I. , Argüello B.
Published: February 2005

The purpose of the present work was to study the feasibility of using hamster oocytes stored at 4°C in M‐2 culture medium for 24 and 48 hours in the evaluation of human sperm fertilizability. A total of 1,394 oocytes were stored for 24 hours and 1,234 were stored for 48 hours. After the storage period all the ooctyes were stained with fluorescein diacetate, proving the physical integrity of the egg plasma membrane. Twenty‐five and 22 semen samples were used to compare their ability to penetrate freshly ovulated and 24‐and 48‐hour‐stored hamster oocytes. Freshly ovulated and 24‐hour‐stored oocytes were penetrated at percentages that in more than 95% of the cases showed no significant differences. The same experiments carried out with oocytes stored for 48 hours showed that in 75% of the cases no significant differences were found. The use of oocytes preserved at 4°C when large numbers of semen samples are to be tested for fertilizability is recommended.
Protamine in the rat, its fate in vivo, and its degradation in vitro by egg homogenate
Authors: Betzalel Moshe , Shalgi Ruth , Moav Boaz
Published: February 2005

Spermatogenesis in the male rat involves chemical and physical transformation of chromatin. There is a shift in the chemical composition of rat testicular sperm chromatin containing histones to nucleoprotamines in the mature sperm. Rat protamine contains a high percentage of arginine. The biosynthesis and kinetics of H3‐arginine incorporation into rat sperm protamine was at its peak in the testis 2 days after intratesticular injection. The specific activity of protamine in the testis was reduced to 50% 5 days after injection. Specific activity of protamine in the epididymis was maximal at 9 days after injection and remained at a similar level for 12 days. The kinetics of H3‐arginine and H3‐thymidine in ejaculated sperm were compared. It was found that maximal specific activity of H3‐arginine (H3‐Arg/No. of sperm) in the spermatozoa was obtained 20–22 days following its intratesticular injection. The peak of H3‐thymidine specific activity (H3‐thymidine/No. of sperm) was obtained 53–59 days following the injection.

H3‐Arg labeled sperm were capacitated in vitro and were allowed to penetrate zona free rat eggs in vitro. The disappearance of the label from the sperm head was observed 1–2 hr after penetration. Protamine labeled in vitro with I125 incubated with extracts of freshly ovulated rat eggs shows that protamine could be degraded by the cytoplasmic extract (10–30%). The proteolytic activity is maximal at pH 7.0–8.0. Cytoplasmic extracts from cumulus cells incubated with I125 rat protamine under identical conditions did not show any significant proteolytic activity. Trypsin degradation of rat protamine shows a different pattern of degradation. It is assumed that the H3‐labeled protamine in the sperm chromatin is removed and probably degraded early after penetration, making possible the remodeling of the sperm chromatin by embryonic histones.
Partial purification and characterization of sperm receptor hydrolase, a cortical granule proteoesterase, from eggs of the sea urchin Strongylocentrotus purpuratus
Authors: Lois Augusto F. , Lackey Debora A. , Carroll Edward J.
Published: February 2005

Sperm receptor hydrolase, one of two classes of cortical granule proteoesterases (E.C.3.4.4.4) was purified approximately 30‐fold with 80% yield from Strongylocentrotus purpuratus cortical granule exudate. Sperm receptor hydrolase preparations were free of vitelline delaminase activity (the other class of cortical granule proteoesterase) and had less than 1% of the starting levels of cortical granule peroxidase and β‐1,3‐glucanohydro‐lase activities. Native polyacrylamide gel electrophoresis coupled with a protease activity stain showed that three proteases were present in the most highly purified preparations of sperm receptor hydrolase. Each of the three proteases has the same molecular weight of 60,000, but different isoelectric points of 2.4, 3.8, and 5.5. The Km value of the mixture of proteases for α‐N‐benzoyl‐L‐arginine ethyl ester as substrate was 263 μM at pH 8.4 and 30°C; the pH dependence of Vm showed a single prototrophic group with a pK of 6.7 and an enthalpy of ionization of 8.6 kcal‐mol−1. The values of these kinetic parameters are consistent with an enzyme‐active site containing histidine. Phenylmethyl sulfonyl fluoride, tosyl lysine chloromethyl ketone, several proteinaceous trypsin inhibitors, and p‐aminobenzamidine inhibited the esterase activity of the proteases. These data suggest that sperm receptor hydrolases are serine proteases.
Effect of in vivo oocyte aging on sperm chromatin decondensation in the golden hamster
Authors: Jedlicki A. , Barros C. , Salgado A. M. , Herrera E.
Published: February 2005

Aged spontaneously activated hamster oocytes recovered from adult females 18 and 24 hours after ovulation were at the pronuclear stage. These oocytes and fresh controls were inseminated in vitro with capacitated hamster spermatozoa and observed with the phase‐contrast microscope. The percentage of fertilization in fresh control oocytes was 98%, as compared to 36% and 18% when the oocytes were recovered 18 and 24 hours after ovulation, respectively. The mean number of sperm decondensations per egg in control oocytes was 10, and in the aged ones it was 0.69 and 0.12 when the oocytes were recovered 18 and 24 hours after ovulation, respectively. When similarly treated oocytes were studied with scanning and transmission electron microscopy, it was found that the degree of gamete membrane fusion was greater than that observed with the phase‐contrast microscope, but that most of the spermatozoa failed to decondense the chromatin. We suggest that parthenogenetic oocytes at the pronuclear stage are in a similar stage of the cell cycle as in fertilized eggs, in which the cytoplasm does not have the ability to decondense the sperm chromatin.
Two simple methods for detecting acrosome‐reacted human sperm
Authors: Cross Nicholas L. , Morales Patricio , Overstreet James W. , Hanson Frederick W.
Published: February 2005

We describe two methods for detecting acrosome reactions of human sperm at the light microscopic level. The techniques include the use of a supravital stain to detect dead sperm in order to differentiate between “physiological” and “degenerative” acrosome reactions. Sperm are incubated with the supravital stain Hoechst 33258 (a fluorescent DNA‐binding dye with limited membrane permeability), washed, suspended in 95% ethanol for fixation and permeabilization, and dried onto slides. The sperm are then labeled either by indirect immunofluorescence using rabbit anti‐human sperm antiserum or with fluoresceinated Pisum sativum agglutinin (PSA). Both probes intensely label the acrosomal region of acrosome‐intact sperm. Electron microscopy revealed the major site of PSA binding to be the acrosomal contents. Acrosome‐reacted sperm have diminished acrosomal labeling by both probes; sperm with nuclei labeled by Hoechst stain are considered nonviable, and are excluded from the assay. Both assays are rapid, give similar results, and detect an increase in acrosome reactions following exposure to the ionophore A23187.
Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′‐dihexyloxacarbocyanine iodide)
Authors: Luttmer Shirley J. , Longo Frank J.
Published: February 2005

We have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′‐dihexyloxacarbocyanine iodide (DIOC6) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae‐free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC6 staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC6 (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC6‐positive organelles. Sea urchin and surf clam sperm stained with DIOC6 fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC6 were less motile than unstained sperm, they were capable of inseminating only zonae‐free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.
Characterization of cAMP‐dependent protein kinase and its endogenous substrate proteins in ram testicular, cauda epididymal, and ejaculated spermatozoa
Authors: Wooten Marie W. , Voglmayr Josef K. , Wrenn Robert W.
Published: February 2005

Mammalian spermatozoa have been shown to possess cAMP‐dependent protein kinase (A‐PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A‐PK, stimulated over a concentration range of 10−9 to 10−5 M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10−6M), testicular spermatozoa had significantly greater amounts of cAMP‐dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP‐40‐soluble protein extracts revealed the presence of two substrate proteins (Mr = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (Mr = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP‐modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.
In vitro fertilization of hamster and human oocytes by microinjection of human sperm
Authors: Lassalle B. , Courtot A. M. , Testart J.
Published: February 2005

In this study, swollen sperm heads were obtained after the injection of human sperm into the perivitelline space of hamster oocytes. The number of injected sperm and the sperm concentration in the preincubation medium were found to have an influcnce on the rate of penetrated hamster oocytes. The optimal injected sperm number was always between five to 12 to obtain 8, 37, and 36% penetration for donors A, B, and C, respectively. The optimal sperm concentration in preincubation medium was between 6 and 22 × 106 sperm/ ml to obtain 16, 47, and 43% penetration for donors A, B, and C, respectively. The rate of polyspermic oocytes was related to the injected sperm number (0, 55, and 100% for one to four, five to 12, and more than 12 injected sperm respectively). Ten human mature oocytes were injected with the sperm from six normal donors. Five fertilized eggs were obtained, and of these four cleaved in in vitro culture.
Distribution by sex of mouse fetuses in the intrauterine position
Authors: Endo A. , Goto T. , Sakai N.
Published: February 2005

We examined whether or not the sex of the fetuses of polytocous animals distributes randomly in the position along the uterine horn in 255 mouse litters. The fetal sex ratio did not differ significantly among the three intrauterine segments (ovarian, middle, and cervical). Based on the number of fetuses examined in this study, it can be stated that even if sex ratio differences exist among the segments, the ratio in individual segments would fall mostly inside the ±0.1 range, when the overall sex ratio (M/(M+F)) is around 0.5.
Spontaneous continuous release of spermatozoa and its predawn surge in male passerine birds
Authors: Quay W. B.
Published: February 2005

Spontaneous, continuous release of morphologically normal spermatozoa occurs in males of species of passerine (order Passeriformes) birds that were examined. It was demonstrated and studied quantitatively in temporarily captive and isolated house sparrows and house finches by means of repetitive cloacal lavages and extraction of excreta. It is suggested that this phenomenon could be exploited to facilitate comparative and quantitative evaluations of release of spermatozoa in relation to diverse environmental, physiological, and social factors.
Seasonal variation in estrous cycling in the mouse: Implications for artificial insemination
Authors: Watson J. Gary , Chaykin Sterling
Published: February 2005

Artificial insemination in the C3HeB/FeJ inbred strain of mice has been shown to be more successful at the middle and end of the calendar year. The reasons are twofold: 1) an increase in the number of normal estrous cycles exhibited by females and 2) an increase in the tightness of the phasing of ovarian and vaginal events. The latter phenomenon was found to be the key to the success of artificial insemination, since it permitted the use of vaginal smears to predict accurately the time females could be expected to ovulate and, therefore, the appropriate time for artificial insemination. Seasonal variations in the frequency of estrous cycling also have been observed in SJL/J and B6D2F1/J females.
Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona‐free hamster ova in vitro
Authors: Clarke R. N. , Johnson L. A.
Published: February 2005

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona‐free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid‐stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at −196°C for 3 days). A highly motile sperm population was isolated by the swim‐up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris‐buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F‐10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome‐reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.
Ultrastructural study of the mature egg of Tethya citrina sarà and melone (porifera, demospongiae)
Authors: Gaino Elda , Burlando Bruno , Buffa Paola , Sarà Michele
Published: February 2005

Tethya citrina is an oviparous demosponge in which eggs are distributed in clumps within the choanosome. The cytoplasm of the mature egg presents a peripheral cortex consisting of a slightly granular layer sandwiched between two densely granular, vesiculated ones. The cortex probably has a specialized, trophic function. Mesohyl bacteria are phagocyted at the egg surface, included in vacuoles, and transferred across the cortical sheath toward the inner cytoplasm. The region of the egg extending between the cortex and the nucleus shows a lacunary system mostly developed beneath the cortical envelope. The noncortical cytoplasm also contains lipid droplets, dense rodlike bodies, and phagosomelike granules. Most of the latter are probably autophagosomes, forming lacunae and supporting autosynthetic vitellogenesis. Rodlike inclusions are probably proteinaceous; they likely originate within the phagosomes and represent the actual yolk material.
Distribution of microtubules during the first meiotic cell division in the mouse oocyte: Effect of taxol
Authors: Rime Hélène , Jessus Catherine , Ozon René
Published: February 2005

Mouse oocytes were stained by antitubulin antibody and by an anti‐MAP1 antibody (JA2) during the first meiotic cell division. At germinal vesicle stage, JA2 antibody exclusively stains the nucleoplasm. When the nuclear envelope breaks down (GVBD) (2–3 h of culture), numerous foci of microtubules appear around the disrupting nuclear envelope; they are decorated by anti‐MAP1 antibody. After 10–12 h of culture, the antigen component detected with anti‐MAP1 antibody is present in the meiotic spindle.

The antimitotic agent taxol (10 μm) induces microtubule formation, mainly at the periphery of the germinal vesicle. At GVBD, taxol provokes the formation of a large microtubular array stained with both antibodies, which is associated with the condensed chromosomes. Furthermore, numerous cytoplasmic asters become visible in the cytoplasm. At metaphases I and II, taxol induces an important enlargement of the metaphase spindles and increases the number of cytasters.
Structural organization of the sperm chromatin in a fern (Scolopendrium vulgare) studied by spreading methods
Authors: Khlifi Samira , Robert Daniel
Published: February 2005

To investigate chromatin organization, we applied the spreading techniques to nuclei isolated from Scolopendrium spermatozoids.

Well‐dispersed chromatin shows three types of fibers: beaded fibers corresponding to a nucleosomal filament with adjacent nucleosomes in close contact, smooth fibers (14 nm in diameter) associated in a complex network, and knobby fibers constituted by local supercoiling of a very thin (4 nm) smooth filament. Along the knobby fibers, beads of variable size are irregularly spaced.

The knobby fibers lie parallel and coalesce in thick bundles. The sperms basic proteins identified by electrophoretic analysis probably promote the supercoiling and the side‐to‐side attachment of the knobby fibers, which are all the more abundant in spread preparations. These results indicate that knobby fibers are probably located in the outer part of the sperm nucleus in which the chromatin is densely packed. As for the nucleosomal and smooth filaments, they may be situated in the inner part.
Intratesticular injection as a method to assess the potential toxicity of various agents and to study mechanisms of normal spermatogenesis
Authors: Russell Lonnie D. , Saxena Nirmal K. , Weber James E.
Published: February 2005

To better understand, to optimize, and to validate the technique of intratesticular (i.t.) injection, several parameters related to i.t. injection were examined. Volumes exceeding 50 μl could be injected i.t.; however, testes frequently became excessively turgid and backflow of injected fluids occurred. Thus, a volume of 50 μl or less was deemed optimal for injection. To determine the rate of distribution of substances throughout the testis, trypan blue was injected i.t. near the caudal pole of the testis, and the movement of dye was monitored. Within 2 min, the dye had spread approximately 1 cm from the site of injection, and in 5 min it had spread twice that distance. In 2 h, the dye had become distributed throughout the testis except at its extreme cranial pole. Seminiferous tubules did not take up dye, indicating that the spread of dye was via peritubular lymphatics. Seminiferous tubule histology appeared virtually unaffected by i.t. injection, even at regions adjacent to the site of injection, when a sterile 26‐gauge or smaller bore needle was utilized. To determine disappearance from the testis, radiolabeled inulin was injected i.t. Half time for absorption was achieved at 1.75 h. Potential vehicles were expolored in which compounds with a variety of physical properties could be injected. Gum tragacanth, normal saline, ethylene glycol, dimethyl sulfoxide (DMSO) mixed 1:1 with normal saline, sesame oil, and propylene glycol were found to be suitable injection vehicles, whereas ethanol, dissolved in normal saline in concentrations as low as 0.5% was found unsuitable. To assess vehicle efficiency, various vehicles were utilized with a known testicular toxin (taxol) and injected into one testis, and the histology was compared with the contralateral testis injected with vehicle alone. All vehicles, found suitable above, allowed dispersion of taxol to influence areas distant from the site of injection. Intratesticular injection assesses the potential of agents to directly affect the testis, and systemic metabolism is avoided. Their rapid spread throughout the lymphatics of the testes allows seminiferous tubules to be exposed to agents in innocuous vehicles more rapidly and in higher concentration than is often possible when using systemic injections.
Outer acrosomal membrane of guinea pig spermatozoa: Isolation and structural characterization
Authors: Olson G. E. , Winfrey V. P. , Winer M. A. , Davenport G. R.
Published: February 2005

We describe a protocol to isolate a highly enriched fraction of outer acrosomal membrane from guinea pig spermatozoa and present new data on the ultrastructure of this membrane domain. Cauda epididymal spermatozoa were suspended into a low ionic strength buffer and subjected to brief homogenization; this stripped the plasma membrane from the spermatozoa and severed the acrosomal apical segment from the spermatozoon. The crescent‐shaped apical segments retained the outer acrosomal membrane and specific components of the acrosomal matrix. Enriched fractions of apical segments were isolated on discontinuous sucrose gradients and the outer acrosomal membrane purified by subsequent centrifugation onto Percoll density gradients. The isolated outer acrosomal membrane did not form vesicles, but instead rolled up into spiral sheets. Both thin section and negatively stained specimens revealed a paracrystalline arrangement of filaments associated with the luminal surface of the membrane. The isolated outer acrosomal membrane revealed a limited number of polypeptides by SDS‐PAGE, and the polypeptide pattern was distinct from the plasma membrane fraction. The isolated acrosomal membranes possessed no oubain sensitive Na+, K+‐ATPase activity, whereas about 20% of the ATPase activity of the plasma membrane enriched fraction was inhibited by oubain. The potential function of the structural differentiations of the outer acrosomal membrane in the membrane fusion events of the acrosome reaction is discussed.
Identification of a male‐specific histocompatibility protein on preimplantation porcine embryos
Authors: White K. L. , Anderson G. B. , Berger T. J. , Bondurant R. H. , Pashen R. L.
Published: February 2005

An indirect immunofluorescence assay was used to detect the presence of male‐specific protein(s) on various stages of preimplantation porcine embryos. Embryos were collected at slaughter from the reproductive tracts of day−2.5, −4, −5, −6, and −8 (day 0 = first day of estrus) sows and gilts. Embryos were placed in medium containing an anti‐male primary antibody, washed, and transferred to culture drops containing a fluorescein isothiocyanate (FITC)‐labeled secondary antibody. Embryos were classified as either fluorescent (H‐Y positive) or nonfluorescent (H‐Y negative), transferred to coded drops, and karyotyped to examine sex chromosomes. A total of 91 eight‐cell to blastocyst stage embryos were evaluated; of these, 46% were classified as fluorescent and 54% as nonfluorescent. Of readable metaphase spreads (65%) from these embryos, 81% (48 of 59, P < 0.005) were correctly sexed by immunological detection of the male‐specific antigen. Although 13 % (2/15)of four‐cell embryos evaluated were classified as fluorescent, the accuracy with which embryos at this stage were sexed by detection of H‐Y antigen was not different from 50%. Fifty percent of eight‐cell embryos were classified as H‐Y positive with 78% of embryos correctly sexed. It was concluded that the eight‐cell embryo is the earliest stage of development for which there is evidence for expression of H‐Y antigen. Detection of the male‐specific protein was difficult at the expanded blastocyst stage.
Impaired spermatogenesis in the finch hybrid L. castaneothorax by L. punctulata: Transmission electron microscopy and genetic analysis
Authors: Swan M. A. , Christidis L.
Published: February 2005

As part of a project to determine whether there is any correlation between the form of hybrid sterility and the genetic relatedness of the parental species, we studied a male intrageneric hybrid between two finch species (Lonchura custaneothorax × L. punctuluta), and compared the ultrastructural basis of hybrid sterility in this species with that reported by Swan [1985] for an intergeneric bird hybrid. In the latter study the sterility appeared to have an autoimmune basis, due to lack of Sertoli‐Sertoli tight junctions. In the hybrid examined in the present study, lanthanum tracing showed that the junctions were tight. There was no testicular immune reaction; the parental species were almost identical in chromosomal constitution, having only a small inversion difference on chromosome 5, and only two structural protein differences could be detected through examination of the variation at 38 protein loci. Nevertheless, the hybrid appeared sterile and had the following ultrastructural testicular features. Intercellular bridges where present were usually abnormal in structure; centrioles in a centriole pair were arranged in parallel. Many spermatocytes and spermatids degenerated and were phagocytosed by Sertoli cells. Some spermatids progressed to mature testicular spermatozoa in sperm bundles, but commonly had multiple (2–4) axonemes or disrupted doublets and accessory fibers. The multiple axonemes present in most spermatids inserted separately into the base of the nucleus and the multiple centrioles were capable of organizing separate neck structures. We conclude that these cytological abnormalities were caused by genic effects and discuss why they appeared to be restricted to the germ line.
Effects of exposing bovine sperm to bovine serum albumin, or freeze‐thawing, on sperm‐bound amidase activity
Authors: Schenk J. L. , Amann R. P.
Published: February 2005

Isolation of a self‐selected population of motile spermatozoa is possible by using a gradient of bovine serum albumin (BSA). We determined if exposure to BSA altered the sperm or if isolated sperm differed from nonisolated cells in terms of motility or activity of sperm‐bound amidase, either before or after subsequent cryopreservation. Exposure of sperm to 6% BSA in egg yolk Tris extender induced changes in the plasma and acrosomal membranes of sperm that resulted in exposure and activation of sperm‐bound amidase (P < .01). In experiment 2, semen extended in egg yolk Tris was cooled to 5°C or layered onto a solution of 6% BSA in extender at 37°C, from which the sperm that had swum into the BSA solution were recovered 2 h later and cooled to 5°C. Sperm in both treatments were cryopreserved. The percentage of progressively motile sperm was determined visually and by track motility. Activity of sperm‐bound amidase exposed to substrate was evaluated. After recovery of sperm from the 6% BSA solution, 81% were progressively motile as compared to 59% in the starting samples (P < .01). However, the amount of exposed sperm‐bound amidase also was greater (P < .05) this was a deleterious change. Immediately after thawing, more (P < .01) sperm were motile in samples of isolated sperm than for nonisolated cells (43 vs 24%), but after incubating the thawed sperm for 1 h at 37°C there was no difference. After freezing and thawing of sperm, amidase activity was higher (P < .05) for the isolated sperm than for nonisolated cells. Thus, isolation of sperm using a 6% BSA gradient increased the proportion of progressively motile sperm, but decreased the percentage of sperm with an intact acrosome, based on measurements of amidase activity.
Sperm chromosome analysis as a new system to test human X‐ and Y‐sperm separation
Authors: Ueda Katsunori , Yanagimachi R.
Published: February 2005

The correlation between the percentage of F‐body‐positive spermatozoa (F‐sperm) and that of Y‐chromosome‐bearing spermatozoa (Y‐sperm) determined by human sperm chromosome analysis was examined after spermatozoa were separated by an albumin column method [Ericsson et al, Nature 246:421–424:1973]. The percentages of F‐sperm were 43.1 ± 4.8 (36.1–48.8) and 45.8 ± 8.3 (35.3–66.5) before and after separtion, respectively. A slight but significant (p < 0.05) increase in F‐sperm after the separation was observed in two semen donors. A total of 570 sperm karyotypes was analyzed and the percentage of Y‐sperm ranged from 36.0 to 59.1 with a mean of 47.3. In one semen donor, the percentage of Y‐sperm (45.6) was significantly (p< 0.05) higher than that of F‐sperm (35.3) after separation. In the remaining nine semen donors, the results obtained by chromosome analysis corresponded well with those obtained by F‐body scoring. The validity of the albumin column method in isolating a Y‐sperm‐rich sperm population is discussed.
Formation of the rupture site in preovulatory hamster follicles: Morphological and morphometric analysis of thinning of the granulosa and thecal layers
Authors: Martin G. G. , Talbot P.
Published: February 2005

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