bluuebunny/crossref_metadata_2025 huggingface dataset:
Maturation of the rat cumulus‐oocyte complex: Structure and function
Authors: Phillips David M. , Dekel Nava
Published: February 2005

The cumulus cells that surround the mammalian oocyte become dispersed following the preovulatory surge of the pituitary gonadotropin, luteinizing hormone (LH). We have examined cumulus‐oocyte complexes of PMSG‐primed immature rats before and at 1, 2, 3, 4, 6, and 8 hr after injection of human chorionic gonadotropin (hCG), which acts on the rat ovary like the pituitary gonadotropin. Associations between projections of the cumulus cells and the oocyte were analyzed in thin sections. We observed that some cumulus projections were greatly enlarged where they associate with the oocyte. These enlarged regions were filled with numerous small vesicles. Gap junctions between cumulus cell projections and the oocytes were small. We quantitated the number and size of gap junctions between cumulus cells. The number of small gap junctions (<1 μM) between cumulus cells did not change significantly over the 8‐hr period after hCG administration. Large gap junctions, however, showed a general downward trend beginning after the third hour post hCG. Light microscopic observations of plastic sections revealed that dispersion of the cumulus oophorus is not observed until after 4 hr post‐hCG, but between 4 and 8 hr after gonadotropin administration the cumulus becomes markedly dispersed. In the majority of the oocytes in these complexes the germinal vesicle (GV) displayed some irregularity in shape at 2 hr post‐hCG, although absence of the GV was not observed until later.

Our observations suggest a new means of communication in the cumulus‐oocyte complex by the vesicle‐filled enlargements of the cumulus cell projections at the oocyte surface. They further indicate that the decrease in metabolic coupling observed in rat cumulus‐oocyte complexes soon after exposure to LH is not associated with a change in number and size of the gap junctions between the cumulus cells. We suggest that it is either the disruption of the gap junctions at the region of contact of the cumulus cell projections with the oocyte surface or the operation of a gating mechanism that blocks the junctional channels without affecting their morphological appearance that is responsible for uncoupling of the oocyte from the cumulus cells.
Expression of the rig gene in mouse oocytes and early embryos
Authors: Taylor Kent D. , Pikó Lajos
Published: February 2005

A clone selected from a two‐cell mouse embryo cDNA library has been sequenced and identified as rig cDNA. The rig gene codes for a highly conserved nuclear protein, which may have a general role in cell growth or replication (Shiga et al.: Proc Natl Acad Sci USA 87:3594, 1990). The quantitative changes in rig mRNA were studied in blot hybridization experiments with total RNA from oocytes and early embryos. The amount and relative abundance of rig mRNA change considerably during early development. There are about 1.6 × 104 rig mRNA molecules in a late growth‐stage oocyte; this number is reduced to about one‐tenth in the ovulated egg but increases about twenty‐fold during cleavage through the blastocyst stage. In F9 embryonal carcinoma cells, the relative abundance of rig mRNA is similar to that in blastocysts (about 0.1% of the mRNA population), but it is about eight‐fold higher in the mouse myeloma cell line MOPC‐104E. The high level of rig mRNA in late growth‐stage oocytes suggests that the rig gene product may be important for cverall transcriptional activity rather than DNA replication and mitosis. Alternatively, the rig protein may be a storage product of oogenesis and have a role in the initiation of development.
Genomic imprinting: Male mice with uniparentally derived sex chromosomes
Authors: Handel Mary Ann , Hunt Patricia A.
Published: February 2005

Although it has been known that there is an X‐chromosome imprinting effect during early embryogenesis in female mammals, it remains unknown if parental origin of the X chromosome has an effect in males. Furthermore, it has not been possible to produce animals with normal sex chromosomes of uniparental origin to further evaluate such imprinting effects. We have devised a breeding scheme to produce male mice, designated XPYP males, in which both the X and Y chromosomes are paternally inherited. To our knowledge, these are the first mammals produced that have a normal sex chromosome constitution but with both sex chromosomes derived from one parent. Development and reproduction in these XPYP males and the sex ratio and chromosome constitution of their offspring appeared normal; thus there is no apparent effect in males of having both sex chromosomes derive from one parent of of having the X chromosome derived from an inappropriate parent. Although we have detected no X‐chromosome imprinting effect in these males, evidence from other sources suggests that the X chromosome is parentally imprinted. Thus detection and definition of an imprint can depend on the assay used.
Oxygen transport to embryos in microdrop cultures
Authors: Baltz Jay M. , Biggers John D.
Published: February 2005

The standard method for culturing small preimplantation mammalian embryos is a system in which they are placed into microdrops of culture medium under oil. Thus the source of oxygen for the embryos lies beyond two liquid phases—medium and oil. If transport of oxygen is not sufficiently rapid to replace that consumed by the embryos, the medium could become depleted of oxygen. Over small distances, the dominant means by which oxygen is transported through liquid is by diffusion. Our calculations show that diffusion alone is sufficient to supply oxygen to mouse embryos; there is virtually no perturbation of the oxygen concentration when there are up to 10 embryos in the drop, and 50 embryos produce a drop in oxygen tension that is not large enough to have a deleterious effect. Furthermore, diffusion is probably not the dominant mechanism by which oxygen is transported to the embryos; on the scale of these microdrops, convection is faster and would serve to mix the drop so that anoxic regions cannot develop. Therefore, we conclude that even a relatively large number of embryos in a culture drop do not significantly deplete oxygen.
Formation of the perinuclear theca in spermatozoa of diverse mammalian species: Relationship of the manchette and multiple band polypeptides
Authors: Longo Frank J. , Cook Susan
Published: February 2005

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm; guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub‐ and post‐acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.
Electroporation of bovine spermatozoa to carry foreign DNA in oocytes
Authors: Gagné Marc B. , Pothier François , Sirard Marc‐André
Published: February 2005

In the present study, electroporation was used to test the ability of spermatozoa to carry foreign DNA into the bovine oocytes. Frozen‐thawed bovine spermatozoa (107/ml) were electroporated using six different combinations of voltge (500, 1,000, or 1,500 V) and capacitance (1 or 25 μ Farads) in the presence of 1 mg/ml of plasmid pRGH527. The portions of plasmids retained by sperm cells after three washings (stable for ten washings) were 4.3, 5.5, 5.1, 6.0, 6.8, and 5.8% for 1 μFarad, 500, 1,000, and 1,500 V and 25 μFarads, 500, 1,000, and 1,500 V, respectively. Nonelectroporated cells have retained only 1% of plasmids. In the same experiment, electroporated spermatozoa were acrosome reacted by treatment with ionophore A23187 to evaluate the fraction of marked plasmids joined at the acrosomal membrane. The results show that 3.5, 5.0, 4.4, 5.0, 6.3, and 4.4% remain tied to the ionophore‐treated sperm. Only 0.7% of plasmid was retained after removal of the acrosome of nonelectroporated cells. Acrosome reaction was not significantly induced by the electrical field (EF) (P < 0.005). EF decrease motility significantly for > 100 V in 0.3 M mannitol (M) and mannitol‐TALP (MT) (1/1) media and ≥500 V (P<0.05) in TALP medium. The retained plasmid rate was compared between TALP medium M and MT media and resulted in a percentage of 1.0, 2.5, 6.5 at 1 μFarads, 100 V, and 0.9, 3.8, and 3.8 at 25 μFarads, 100 V in TALP, MT, and M medium, respectively. Sperm cells electroporated at 1 μFarad, 500 or 1,000 V, 25 μFarad, 500 V or 1,000 in TALP medium hold plasmids in proportion of 5.2, 5.4, 7.4, and 6.0%. Electroporation above 100 V in M and MT killed the cells. In a part of this experiment, spermatozoa electroporated in the presence of radiolabeled plasmids have been treated with DNase I and results revealed that 35, 28, 54, 58, and 3% of marked DNA remains in sperm cells following digestion after electroporation in TALP (1,000 V, 1 and 25 μFarads), M medium (100 V, 1 and 25 μFarads), and control, respectively. Using in vitro matured bovine oocytes, the electroporation conditions were correlated with the fertilization rate (85% for control and 55% for electroporated spermatozoa). Autoradiography of embryos following fertilization indicated the presence of plasmids in the cytoplasm and in the zona pellucida. Finally, the use of Polymerase Chain Reaction (PCR) revealed the presence of plasmids in blastocyst cells in 12% for control (n=67) and 22, 17, and 19% for electroporated spermatozoa in TALP (n=188,25μFarads, 1,000 V), MT (n=29, 25 μFarads, 100 V) and M (n=21, 25 μFarads, 100 V), respectively. In conclusion, our results indicate A) that electroporation results in an increase absorption of DNA by the spermatozoa, B) that the male gametes can carry foreing DNA in oocytes at fertilisation, and C) that an increased fraction of day 5 embryos is showing a positive response for the inserted gene with the polymerase chain reaction.
Purification and preliminary characterization of guinea pig testicular proteinase inhibitors
Authors: Falase E. A. O. , Storey B. T. , Teuscher C.
Published: February 2005

Three guinea pig testicular, low‐molecular‐weight, acid‐stable inhibitors specific for typsin‐like proteinases were isolated, purified, and characterized. The procedure comprised acid extraction of testicular acetone powder, pH precipitation of the extract, gel filtration of the supernatant on Sephadex G‐100 and G‐50, ion‐exchange chromatography on SP‐Sephadex, followed by QAE‐Sephadex. Final purification was by rechromatography on Sephadex G‐50 superfine gel. The three proteinase inhibitors were labeled A, B, and Cnb, the latter to denote nonbinding of Cnb to the QAE‐Sephadex. Components A and Cnb showed competitive, whereas B showed noncompetitive, inhibition against trypsin. All three inhibitors were active against trypsin but were ineffective against chymotrypsin. The inhibition constants, Ki, were obtained using trypsin‐catalyzed hydrolysis of the N‐benzyloxycarbonyl‐L‐arginyl amide of 7‐amino‐4‐trifluoro‐methylcoumarin (CBZ‐Arg‐AFC) at pH 8.0. The values were calculated to be, for A, 1.5 × 10−8 M; for B, 1.5 × 10−8 M; and, for Cnb, 2.2 × 10−7 M. The Ki values calculated from inhibition of trypsin‐catalyzed hydrolysis of the active site titrant 4‐methylumbelliferyl‐p‐guanidinobenzoate (MUGB) using Easson‐Stedman plots were, for A, 7.7 × 10−9 M; for B, 6.7 × 10−9 M; and, for Cnb, 1.4 × 10−7 M. The MrS as determined by active site titration with MUGB were A, 11.2 kDa; B, 10.5 kDa; Cnb, 17.0 kDa. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gave Mr values for A of 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in Mr values for A and 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in Mr values for B indicates that it may function as a dimer or trimer in the active state.
3H‐uridine incorporation in early porcine embryos
Authors: Freitag M. , Döpke H. H. , Niemann H. , Elsaesser F.
Published: February 2005

The present study investigated the ontogeny of 3H‐uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two‐cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 μM 3H‐uridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. No RNA synthesis was detected at the two‐cell stage. From the four‐cell to the morula stage, 3H‐uridine incorporation per embryo increased about ninefold (P < 0.001) in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. On a per cell basis, 3H‐uridine incorporation was not different from the four‐cell stage up to the zona pellucida‐intact blastocyst and amounted to 0.29–0.37 fmol 3H‐uridine incorporation/cell/3 hr. In hatched blastocyst, 3H‐uridine incorporation per blastomere was increased (P < 0.01 compared with younger stages) and amounted to 0.86 fmol 3H‐uridine incorporation/cell/3 hr. It is concluded that 1) the rate of uridine incorporation depends on the cell stage in zona pellucida‐intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages.
Analysis of proteins synthesized by 9.5 day mouse embryos: Determination of cardiac and noncardiac proteins
Authors: Van Keuren Margaret L. , Iacob Ruxandra A. , Kurnit David M.
Published: February 2005

To catalog polypeptides that were specific to developing hearts, we separated 35S‐methionine‐labeled 9.5 day mouse embyros into cardiac and noncardiac (carcass) components. Two‐dimensional gels were then used to analyze the polypeptides synthesized in these two fractions. As a result, we were able to distinguish polypeptides that were specific to or increased in the heart as well as those polypeptides that were specific to or inceased in the embryo minus the dissected heart. Using this analysis, there were two polypeptides that were cardiac‐specific and 17 that were expressed at increased levels by at least twofold in the heart. The cardiac‐specific polypeptides may be used in further studies to identify early cardiac tissue. Conversely, there were 26 polypeptides unique to noncardiac structures and an additional 15 that were increased in the carcass more than twofold relative to the heart. The noncardiac‐specific polypeptides may be used to define contamination of putative cardiac tissue with noncardiac material. Two of the polypeptides expressed more abundantly in the carcass appeared to correspond to known proteins in the mouse fibroblast database, cyclin and tropomyosin 4. Thus the heart at 9.5 days of murine development can be distinguished readily from the remainder of the embryonic mouse both macroscopically and on two‐dimensional gels.
Anucleate fragments of parthenogenetic eggs and of maturing oocytes contain complementary factors required for development of a male pronucleus
Authors: Borsuk Ewa
Published: February 2005

Eight‐h‐old anucleate fragments of artificially activated mouse oocytes produced within 30 min after activation were fused with anucleate fragments of ovarian oocytes bisected at the GV stage and matured in vitro for 4–5 h. The resulting cybrids were inseminated soon after fusion and fixed 3–4 h or 14 h later. After 3–4 h of culture most of the sperm nuclei underwent decondensation. Early male pronuclei were observed sporadically. After overnight culture small or fully formed male pronuclei were present in monospermic and dispermic cybrids. The abortive nuclei, designated as stage lla (earlier described by Borsuk and Tarkowski, 1989) were also observed and their occurrence was strongly correlated with the rate of polyspermy. In unfused (control) anucleate fragments of maturing and activated oocytes inseminated at the same time as the cybrids, male pronuclei were never observed. These results show that the cytoplasms of activated eggs and of maturing oocytes contain complementary factors required for the transformation of the sperm nucleus into the male pronucleus. The former has lost the capability to break down the sperm nucleus envelope but contains factors required for pronuclear growth. The latter is able only to initiate the process of transformation, i.e., to break down the nuclear envelope and decondense the denuded sperm chromatin. However, the activity of the factor responsible for nuclear envelope breakdown is limited because in cybrids penetrated by more than two spermatozoa, transformation of sperm nuclei was usually abnormal.
Cytochalasin D treatment induces meiotic resumption in follicular sheep oocytes
Authors: De Smedt V. , Szöllösi D.
Published: February 2005

Ovine cumulus‐enclosed oocytes collected from antral follicles (3–5 mm in diameter) were cultured in vitro with 2 ± 106 granulosa cell/ml in the presence or absence of gonadotropins or in the presence of cytochalasin D (CD). The maturation rate was assessed after 24 h of culture. In the control group, in the presence of gonadotropins (follicle‐stimulating hormone‐luteinizing hormone (FSH‐LH; 10 μg/ml) 100% of the oocytes reached metaphase II. Whereas intercellular junctions were no longer present after 6–7 h of culture, germinal vesicle breakdown (GVBD) occurred by the same time. In contrast, in the absence of gonadotropin, the majority of the oocytes (59%) remained blocked in GV stage. The inhibition exerted by the granulosa cells on meiotic resumption was overcome when the cumulus‐oocyte complexes (COCs) were incubated in CD (5 μg/ml) for 6 h at the beginning of the culture. Under these conditions, 85% of the oocytes matured with extrusion of the first polar body. Cytological analysis by cytofluorescence (NBD phallacidin) and electron microscopy showed that, after 6 h of treatment, CD provoked a redistribution of the microfilaments, mainly in the cumulus cells and to a lesser extent in the oocyte cortex. Intercellular junctions disappeared concomitantly with a significant decrease of the intercellular transport of tritiated uridine. The initiation of GVBD occurred at the same time. These results indicate that the resumption of meiosis was correlated with a loss of both junctional complexes (intermediate and gap junctions) between the cumulus cells and the oocyte.
Role of the extracellular matrix in tissue‐specific gene expression in the sea urchin embryo
Authors: Benson Steve , Rawson Robert , Killian Christopher , Wilt Fred
Published: February 2005

The role of extracellular matrix (ECM) in the differentiation of tissue types was examined in embryos of Strongylocentrotus purpuratus. We have examined the expression of various tissue‐specific molecular markers after disrupting the ECM by culturing embryos in the presence of β‐aminoproprionitrile fumarate (BAPN), which disrupts collagen deposition, and β‐D‐xyloside, which disrupts proteoglycan metabolism. The markers examined included accumulation of primary mesenchyme‐specific mRNA (SM 50); and aboral ectoderm‐specific mRNA (Spec 1); and a gut‐specific enzyme, alkaline phosphatase. Treatment with BAPN or β‐D‐xyloside results in developmental arrest at the mesenchyme blastula stage. Although spicule formation is inhibited, the accumulation of SM 50 transcripts and the synthesis of most of the prominent spicule matrix proteins is similar to that of control embryos. Spec 1 mRNA, in contrast, while accumulating to a significant extent when collagen and proteoglycan metabolism is disrupted, does accumulate to a level somewhat lower than that seen in control embryos. Additionally, the postgastrula rise in gut‐specific alkaline phosphatase is reversibly inhibited by BAPN and xyloside treatment. These results demonstrate a differential effect of the ECM on expression of tissue‐specific molecular markers.
Unique 3′‐untranslated sequence of insulin‐like growth factor‐I isolated from human placenta
Authors: Daimon M. , Wang C.‐Y. , Johnson T. R. , Ilan Joseph , Ilan Judith
Published: February 2005

A cDNA clone of 525 bp corresponding to the 3′‐untranslated region of insulin‐like growth factor‐I was isolated from a human placenta library. The sequence of this clone extended 200 nucleotides downstream from the previously reported 3′‐end of IGF‐IA cDNA, indicating the existence of IGF‐IA transcripts having an even larger 3′‐untranslated region. By using this clone for RNA transfer blot hybridization, it was shown that this longer 3′‐untranslated region is included in the 7.5‐ and 5.0‐kb transcripts, but not in the 1.1‐ and 0.9‐kb transcripts. It is also apparent that transcripts bearing the extended 3′‐untranslated sequence are highly expressed in human placenta.
Biochemistry of seminal secretions of the crab Scylla serrata with reference to sperm metabolism and storage in the female
Authors: Jeyalectumie C. , Subramoniam T.
Published: February 2005

Biochemical studies on the male reproductive tissues and seminal secretions have been made with reference to sperm metabolism and different stages of maturity in the crab Scylla serrata. The results reveal that the seminal plasma and spermatophores are rich in protein, carbohydrate, and lipid. In general, organic components of spermatophores are considerably higher than those of seminal plasma. Enzyme studies show that the succinate dehydrogenase (SDH) activity is very low, whereas fumarate reductase (FR) and lactate dehydrogenase (LDH) exhibit high activity. Electrophoretic studies on LDH show that, in addition to the occurrence of a sperm‐specific fraction, LDHx, the M‐type subunits are predominant in the mature spermatophores. These results from enzyme studies suggest that sperm metabolism is mainly anaerobic, utilizing the carbohydrates as substrates. The results for maturational changes reveal that the male reproductive tissues and their secretions contain lesser quantity of organic components in the immature crabs; as the maturity proceeds, there is not only concentration of organic substances but also an increase in the size of spermatophores. The concentration of biochemical constituents is highest in the proximal vas deferens (PVD), suggesting that the granular seminal plasma as well as the sperm‐agglutinating substance and spermatophoric wall are secreted in this region. The spermatheca of the unmated female crabs are poor in organic constitutents. After mating, their contents are enriched by organic substances derived from contributions of the seminal substances. During sperm storage in the spermatheca, only the carbohydrates decline steeply. A low activity of SDH, but a moderate level of LDH and a high level of FR activity, is recorded in the spermathecal content of mated crabs, providing further evidence for anaerobic metabolism of sperm during storage in female. A sharp fall in the stored carbohydrates constitutes further evidence in this regard.
Effect of cell‐free synchronous uterine flushings and microsurgery on the development of porcine embryos in vitro
Authors: Martin M. J. , Cantley T. C. , Flowers W. L. , Day B. N.
Published: February 2005

Experiment I was designed to determine if cell‐free synchronous uterine flushings contain an embryotoxic substance that is normally screened by the intact zona pellucida. Sixty 4‐cell embryos were allocated to three treatment groups: (1) control embryos (n = 20) were cultured in Modified Kreb's Ringer Bicarbonate medium + 10% bovine calf serum (mKRB‐BCS), (2) UF embryos (n = 20) were cultured in 80% mKRB‐BCS + 20% sterile dialyzed uterine flushings (UF), (3) MicroUF embryos (n = 20) received a microsurgical incision in the zona pellucida and were cultured in 80% mKRB‐BCS + 20% UF. Following 72 h in culture at 37°C under a 90% N2, 5% CO2, and 5% O2 atmosphere, the number of nuclei/embryo and the incidence of protrusion of the trophoblast through the zona pellucida (PTZ) were recorded. Addition of UF had no effect on embryo development. A greater (P > .005) proportion of MicroUF embryos exhibited PTZ as compared to UF and control embryos. Experiment II was devised to further characterize the occurrence of PTZ in Micro porcine embryos. Thirty‐three 4‐ to 10‐cell embryos and 14 morulae were distributed across two treatments: (1) control embryos (n = 16 and 6, respectively) were cultured as described in Experiment I; and (2) micro embryos were treated similarly to MicroUF embryos in Experiment I but were cultured in mKRB‐BCS only. At the onset of PTZ, embryos were immediately fixed and examined. The proportion of embryos exhibiting PTZ was greater (P < .007) for Micro versus control embryos. Both the mean time to PTZ and the number of nuclei present were greater for blastocysts cultured from control as opposed to Micro morulae (P < .0001 and P < .0002, respectively). These results indicate that (a) cell‐free synchronous uterine flushings are not detrimental to embryos with incised zonae and (b) microsurgery increases the incidence of and reduces the time to protrusion of the embryo through the zona pellucida in cultured embryos.
Presence of follicular fluid in the porcine oviduct and its contribution to the acrosome reaction
Authors: Hansen C. , Srikandakumar A. , Downey B. R.
Published: February 2005

Two experiments were conducted to measure the quantity of follicular fluid entering the porcine oviduct following ovulation and to establish its influence on the sperm acrosome reaction in vivo. Prepubertal gilts treated with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) were used in both experiments. In experiment 1, each of 64 gilts was assigned at random to one of four treatment groups (n = 16 per group): I (preovulatory), surgery 38 hr post‐hCG; II (ovulatory), (surgery) 42 hr post‐hCG; III (postovulatory), surgery 46 hr post‐hCG; IV (ovulation blocked), surgery 46 hr post‐hCG but also treated with indomethacin (INDO) at 24 hr. At surgery, both follicular and oviductal fluid were collected for determination of volume and progesterone (P4) concentration. In experiment 2, sperm were recovered surgically from the uterine horn, isthmus, and ampulla of gilts at 46 hr post‐hCG either (1) inseminated and non‐INDO‐treated controls (n = 5) or (2) inseminated and INDO‐treated at 24 hr (n = 4). Using P4 as a marker, it was calculated that only 0.51% ± 0.10% of the available follicular fluid was present in the oviduct near the time of ovulation and that this amount had decreased 10–12‐fold 4 hr later. Mean sperm concentration at 46 hr post‐hCG was higher in the uterine horn than in the other two regions (P < 0.05) but the percentage of acrosome‐reacted sperm was greater in the ampulla (P < 0.05). The ampullae of ovulating gilts, i.e., containing follicular fluid, had a greater percentage of acrosome‐reacted sperm than those in which ovulation was blocked by INDO (P < 0.05). The results of these experiments suggest that only a small amount of follicular fluid reaches the oviduct at ovulation and that this fluid has a stimulatory, but probably nonessential, effect on the acrosome reaction.
JunD mRNA expression differs from c‐jun and junB mRNA expression during male germinal cell differentiation
Authors: Alcivar Acacia A. , Hake Laura E. , Kwon Yunhee K. , Hecht Norman B.
Published: February 2005

The members of the jun family of protooncogenes (junB, c‐jun, and junD) share a high degree of sequence homology and function as transcriptional regulators. Here we compare the pattern of junD mRNA expression during spermatogenesis to that of junB and c‐jun (Alcivar et al.: J Biol Chem 265:20160–20165, 1990). junD transcripts are present at high levels in total RNA obtained from both prepuberal and adult intact testes, with the highest levels at stages containing predominantly premeiotic and postmeiotic germ cells. Analyses of cells isolated from testes of 8‐day‐old mice indicate that the level of the 1.8 kb junD mRNA is higher in type B spermatogonia than in type A spermatogonia. In testes of 17‐day‐old mice, the highest junD mRNA levels are detected in preleptotene spermatocytes compared to leptotene/zygotene and prepuberal pachytene spermatocytes. In cells from adult testes, the junD mRNA levels are higher in postmeiotic round spermatids and residual bodies/cytoplasts than in meiotic pachytene spermatocytes. An additional junD transcript of about 1.6 kb is detected in postmeiotic cells. Analyses of polysomal and nonpolysomal RNAs prepared from isolated testicular cells indicate that in early meiotic cell types the junD transcript is more efficiently loaded onto polysomes than in later cell types. In summary, the pattern of expression of junD differs from that of junB and c‐jun during spermatogenesis most notably in that 1) junD mRNA levels do not increase following dissociation of testicular cells and 2) in contrast to the nearly undetectable levels of junB and c‐jun mRNAs in adult postmeiotic testicular cells, high levels of junD mRNAs are seen. The differential expression of these three jun transcripts during development of the male gonad suggests a complex transcriptional regulatory role for the jun family of protooncogenes during spermatogenesis.
Studies on murine embryo‐derived platelet‐activating factor (EPAF)
Authors: Adamson L. M. , Podsiadly B. , Smart Y. C. , Stanger J. D. , Roberts T. K.
Published: February 2005

Studies were carried out using the splenectomized mouse bioassay (SMB) to investigate the nature of embryo‐derived platelet‐activating factor (EPAF) and its relationship to synthetic platelet activating factor (PAF). While both C16‐PAF and embryo conditioned media (ECM) induced a significant platelet decline in the SMB at 15 min postinjection, C18‐PAF induced a similar effect at 30 min postinjection. The degree of EPAF activity in ECM was not altered with increasing embryo number from 2 to 40/ml of media. In contrast, PAF (C16/C18 mixture) induced a linear increase in activity with increasing concentration, leading to lethal effects at high concentrations. While EPAF activity was not significantly altered when ECM was diluted 1/1,000, PAF activity was abolished at 1/10 dilution. EPAF in ECM was not inactivated by mouse plasma; however, lipid exracted ECM, like PAF, underwent rapid inactivation in the presence of plasma. Aggregometer studies using horse platelets showed that ECM and lipid‐extracted ECM were unable to induce platelet aggregation, while thin‐layer chromatography (TLC) purified ECM (Rf 0.23) successfully aggregated horse platelets in vitro. Results suggested that EPAF and PAF are not homologous. EPAF might consist of PAF bound to a regulatory carrier molecule and appears to be associated with EPAF‐inhibitor substance(s) in ECM.
Reduction of embryotoxicity by protein in embryo culture media
Authors: Flood Larry P. , Shirley Barbara
Published: February 2005

Experiments tested the hypothesis that one role of protein in embryo culture media is protection of embryos against potentially embryotoxic substances in the media. Mouse embryos were cultured in modified Krebs–Ringer bicarbonate medium and in modified Tyrode's medium, aliquots of which were supplemented with 4 mg/ml of the protein bovine serum albumin (BSA), while other aliquots were left protein free. The media were prepared using water samples that differed in purity, as reflected by differences in conductivity, with tap water being least pure (and considered to have the greatest potential for being embryotoxic) and water that had been purified by reverse osmosis, Milli‐Q filtration, and triple distillation being most pure. Embryos were placed in the media while in the two‐cell stage of development and their development was assessed after 24, 48, and 72 hr of culture. Rate of embryo development in BSA‐supplemented media was greater than that in protein‐free media only when the media was greater than that in protein‐free media only when the media were prepared with the least purified water samples. Because these water samples would have contained substances not contained in media prepared with purer water, or would have contained the substances in higher concentrations, the data supported the hypothesis that protein can protect embryos during culture by negating effects of embryotoxic substances in the media.
Sequence requirements for embryonic transcriptional activation of a gastrula‐specific actin gene in Xenopus laevis
Authors: Brennan Sean M.
Published: February 2005

Cytoskeletal actin genes undergo developmentally timed transcriptional activation at the gastrula stage of embryonic development in the amphibain Xenopus laevis. To study the regulation of this process, a molecularly marked cloned actin gene has been introduced into living embryos by microinjection, and levels of its transcripts (which are distinct from endogenous actin message) have been measured by RNase protection. In vitro mutagenesis of the marked gene, followed by microinjection and transcriptional analysis of various mutants, has been used to search for gene sequences that participate in accurate transcriptional initiation and developmental control. Deletion mutants containing only 90 nucleotides of upstream sequence undergo correct developmental regulation, while deletion to –33 prevents normal activation of the gene. In the presence of sufficient upstream sequence, an actin–globin fusion gene, containing only 564 nucleotides downstream of the actin gene transcription startsite, is correctly activated. Taken together, these results imply that all sequences necessary for correct temporal regulation reside between –90 and +564 nucleotides, with respect to the transcriptional start site of the actin gene. They further suggest that developmental activation of actin gene transcription may involve either (1) interaction of non‐DNA binding proteins with basal transcription factors, or (2) the concerted action of ubiquitous promoter‐binding factors and factors that interact with downstream regulatory regions.
Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo
Authors: Brazolot C. L. , Petitte J. N. , Etches R. J. , Gibbins A. M. Verrinder
Published: February 2005

Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli β‐galactosidase (β‐gal). A reporter construct (phspPTlacZpA) containing a mouse heat‐shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 μg LipofectinTM (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55‐3.1 μg DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn2+‐inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken β‐actin (pmiwZ) or cytomegalovirus (pMaori3) promoters.

Endogenous chicken β‐gal and transferred bacterial β‐gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6–12 h with 150 μM ZnCl2. Bacterial β‐gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have b̃‐gal activity 16–24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct. Plated PCFs were lipofected as well, with stable incorporation of the gene construct indicated in 10% of positive events.

Lipofected CBCs were injected into the subgerminal cavity of stage X (Eyal‐Giladi and Kochav, 1976) chicken embryos in a manner that has been shown to produce somatic and germline chimeras for untreated CBCs (Petitte et al., 1990). After 65 h of incubation, cells expressing β‐gal activity were observed in the prosencephalon, head ectoderm, and ventricle of the heart of a stage 11 (Hamburger and Hamilton, 1951) embryo. In other cases, bacterial β‐gal activity was detected extraembryonically, both in individual cells and in foci of expressing cells, 24, 48, and 65 h after injection. Few, if any, single expressing cells and no foci were detected following injection of the LipofectinTM:DNA mixture directly into the embryo.

Refinement of these procedures could contribute to the development of transgenic poultry, without reliance on retroviral vectors for DNA transmission or incorporation.
Effects of maturation and co‐culture treatments on the developmental capacity of early bovine embryos
Authors: Wiemer Klaus E. , Watson Andrew J. , Polanski Voyteck , McKenna Ann I. , Fick Gordon H. , Schultz Gilbert A.
Published: February 2005

A total of 901 cumulus–oocyte complexes (COCs) were collected from bovine ovaries obtained at a local abattoir. COCs randomly assigned to Treatment I (n=451), were cultured in TCM‐199 + 10% fetal bovine serum (FBS) and hormones, while oocytes in Treatment II (n=450) were cultured in TCM‐199 + 20% estrous cow serum (ECS). Assessment of maturation revealed that 91.3% (42/46) of oocytes in Treatment I had reached metaphase II of meiosis, which was greater (P <0.05) than the 73.3% (33/45) in Treatment II. Following in vitro fertilization, 203 oocytes from Treatment I were co‐cultured on bovine granulosa cells (Treatment IA) while the remaining 202 oocytes were co‐cultured on bovine oviductal cells (Treatment IB). Similarly, 203 oocytes from Treatment II were co‐cultured on granulosa cells (Treatment IIA) or oviductal cells (Treatment IIB, n = 202). Co‐culture was maintained for 8 days. The proportion of cleaved zygotes was higher (P <0.05) in Treatment IB (86.6%) compared to Treatments IA (78.8%), IIA (58.1%), and IIB (64.8%). The proportion of cleaved zygotes that progressed beyond the 16‐cell stage was also greater (P <0.001) in Treatment IB (71.4%) compared to Treatments IA (50.0%), IIA (35.4%) and IIB (55.8%). Treatment IB also produced the highest proportion of blastocysts (P<0.0001) (41.1%) versus 24.6% (IA), 11.3% (IIA) and 18.3% (IIB). The proportion of day 6 morulae that progressed to form day 8 blastocysts was similar for both co‐culture treatments (IA, 70.1%; IB 70.2%; IIA, 51.5%; IIB 50.8%) and varied only between in vitro maturation groups. Results indicate that the combination of in vitro maturation in TCM‐99 + 10% FBS and hormones followed by co‐culture on oviductal monolayers is a superior system for the production of early bovine embryos.
Cytochemical evidence of an organized microtubular cytoskeleton in Xenopus laevis oocytes: Involvement in the segregation of mitochondrial populations
Authors: Tourte Monique , Besse Colette , Mounolou Jean‐Claude
Published: February 2005

An organized microtubular cytoskeleton was discovered in the cytoplasm of Xenopus laevis oocytes. The microtubules were observed in 10‐ to 30‐m̈m cryostat sections by indirect immunoperoxidase labeling using an antibody to tubulin. A gradual extraction of cells with a nonionic detergent was essential for good penetration of the antibody into the cells. In the cytoplasm of all previtellogenic oocytes, a dense network of criss‐crossed long microtubules was associated in a basket‐like structure surrounding the mitochondrial mass. At the beginning of vitellogenesis, the network meshes enlarged, while clusters of mitochondria migrated, in close association with microtubule bundles. At the beginning of vitellogenesis, the reorganization of the microtubular network, mostly in the vegetal hemisphere, occurred during the segregation of the mitochondrial populations. Reorganization is characterized by (1) a temporary enlargement of the network and close association of mitochondrial clusters with microtubular bundles, and (2) a progressive organization of a ring‐shaped microtubular structure in the crown elaboration area. It is hypothesized that these modifications of the microtubular cytoskeleton contribute to the maintenance of cell shape and the polarized organization of the cell.
Equine oocyte in vitro maturation: Influences of sera, time, and hormones
Authors: Willis P. , Caudle A. B. , Fayrer‐Hosken A.
Published: February 2005

Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 μg/ml), equine luteinizing hormone (eLH; 100 μg/ml), bovine follicle‐stimulating hormone (FSH; 5 μg/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P < 0.05) increased by MS, MSO, and FCS as compared with the control (BSA). In vitro culture for either 15 or 32 hr significantly (P < 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P < 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.
Detection of a soluble acrosome reaction‐inducing factor, different from serum albumin, associated with the ovulated egg‐cumulus complex
Authors: Boatman Dorothy E. , Robbins Robert S.
Published: February 2005

Soluble extracts of the ovulated hamster egg‐cumulus complex (ECC) were tested on capacitated sperm for activity in inducing the physiological acrosome reaction (AR). Evidence for occurrence of the physiological AR included enhanced sperm penetration of intact homologous zonae pellucidae as well as induction of AR in nonattached and in zona‐bound sperm following a brief coincubation with test compound. Since hamster serum albumin, a major protein of hamster body fluids, also induces spontaneous ARs under certain conditions, it was used as one of the comparators for the acrosome reaction inducing factor (ARIF; Westrick et al., Biol Reprod 32 [Suppl 1]. 213, 1985) activity in the ECC. Sperm exposure to concentrations of the soluble ECC extract ranging from 0.04 to 0.2 mg protein/ml significantly increased penetration of salt‐stored zonae by 36%, mean numbers of penetrating sperm by 90%, ARs in nonattached sperm by 65%, and ARs in zona‐bound sperm by 102%. Hamster serum albumin added after completion of capacitation had no significant effect on these parameters. We conclude that (1) the ovulated ECC contains a soluble ARIF that augments zona‐induced ARs and sperm penetration and (2) the ARIF is not serum albumin.
Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro
Authors: Yoshida Mitsutoshi , Ishigaki Koji , Pursel Vernon G.
Published: February 2005

The present study was carried out to examine the effect of maturation media on male pronucleus formation of pig oocyte matured and fertilized in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughter house were cultured (36 h) in three different media (mTCM‐199, Waymouth MB 752/1, and mTLP‐PVA), fertilized in vitro, and assessed for nuclear maturation and male pronucleus formation. The addition of 10% (v/v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (P < 0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 752/1 with or without pFF than in oocytes matured in the other two media (P < 0.01). In experiment 2, the addition of cysteine (the same concentration as in Waymouth medium, 0.57 mM), to mTLP‐PVA significantly increased the rate of male pronucleus formation of pig oocytes compared with the control (P < 0.01). The results indicate that the composition of maturation medium affects the ability of pig oocytes to form male pronuclei following sperm penetration; media containing a high concentration of cysteine (possibly as a substrate of glutathione), such as Waymouth MB 752/1, can remarkably promote this ability.