bluuebunny/crossref_metadata_2025 huggingface dataset:
Bovine embryos produce a urokinase‐type plasminogen activator
Authors: Berg Debra A. , Menino Alfred R.
Published: February 2005

The type of plasminogen activator (PA) secreted by bovine embryos was identified. Day 12–14 embryos were collected from estrus‐synchronized, superovulated, and naturally mated crossbred beef cows. Embryos were left intact (E) or microdissected into component embryonic discs (ED) and trophoblastic vesicles (TV). Intact embryos, ED, and TV were pre‐cultured for 2 days in Minimum Essential Medium Alpha (MEMα) with 10% heat‐inactivated fetal calf serum, washed in serum‐free MEMα, and cultured individually for 5 days in 50 μl microdrops of MEMα with 15 mg/ml bovine serum albumin. At 24 hr intervals, E, ED, and TV were observed for tissue morphology and transferred to fresh microdrops, and medium was recovered and frozen at −20°C. At the end of culture, blastocoelic fluid (BF) and embryonic tissues were recovered and frozen at −20°C. Plasminogen activator concentrations in medium, tissues, and BF were determined by using a caseinolytic assay. Antibodies to urokinase‐type PA (anti‐uPA) and tissue‐type PA (anti‐tPA), and the urokinase inhibitor, amiloride (AMR), were used to identify the type of PA produced by bovine embryonic tissues. Intact embryos and TV released more PA (P < 0.05) than ED, and tissues exhibiting expanded blastocoels released less PA (P < 0.05) than tissues with collapsed blastocoels. Blastocoelic fluid from TV exhibited more PA (P < 0.05) activity than from ED. Treatment with anti‐uPA decreased PA activity (P < 0.05) in pooled medium and tissues from E compared to treatment with nonspecific immunoglobulins and anti‐tPA. Amiloride completely eliminated PA activity (P < 0.05) in tissues, medium, and BF. These results suggest that day 12–14 bovine embryos secrete a urokinase‐type PA.
A guanine nucleotide‐binding regulatory protein in human sperm mediates acrosomal exocytosis induced by the human zona pellucida
Authors: Lee Michael A. , Check Jerome H. , Kopf Gregory S.
Published: February 2005

Guanine nucleotide‐binding regulatory proteins play key intermediary roles in regulating zona pellucida‐mediated acrosomal exocytosis in mouse and bull sperm. Since human sperm possess a Gi‐like protein and undergo the acrosome reaction in response to the human zona pellucida, we investigated whether this G protein plays a regulatory role in this exocytotic process. Zonae pellucidae isolated from eggs that had been inseminated but had shown no signs of fertilization after retrieval for in vitro fertilization and embryo transfer were pooled into groups of ≥50 in order to reduce variability in biological responses due to the possible presence of ZP that had undergone modifications associated with the polyspermy block. Acid‐solubilized zonae pellucidae were incubated with capacitated sperm, and the sperm then assessed for the acrosome reaction using both the P. sativum agglutinin and chlortetracycline fluorescence assays; both assays gave similar results. Sperm incubated with solubilized zonae pellucidae at a final concentration of 2, 4, or 6 ZP/μl underwent acrosomal exocytosis to a similar extent as compared with A‐23187. Sperm were incubated with 1 μg/ml pertussis toxin during capacitation to functionally inactivate the Gi‐like protein. Pertussis toxin treatment of sperm did not affect sperm motility and the ability of the cells to bind to structurally intact zonae pellucidae. Pertussis toxin, however, completely inhibited the percentage acrosome reactions induced by solubilized zonae pellucidae. By contrast, the A‐23187‐induced acrosome reaction was insensitive to PT treatment. Pertussis toxin inhibition of the zona pellucida‐induced acrosome reaction occurred in a concentration‐dependent manner with maximal effects observed at 100 ng/ml PT. These data suggest that the pertussis toxin‐sensitive Gi‐like protein in human sperm plays an important regulatory role in the acrosome reaction induced by the human zona pellucida.
Cortical granule complements in human oocytes undergoing partial zona dissection
Authors: Lanzendorf Susan E. , Kazer Ralph R. , Patton Phillip E. , Wolf Don P.
Published: February 2005

This study was performed to evaluate the effects of mechanical stimulation and sucrose treatment on the oocyte activation process. Fresh and aged human oocytes were exposed to sucrose and zonae were dissected with microneedles before fixation and quantitative analysis of cortical granules by transmission electron microscopy. Examination of the mean number of cortical granules/analyzed segment revealed no significant differences between control oocytes or oocytes treated with sucrose or sucrose treatment followed by zona dissection. A significant decline in the number of cortical granules/segment was observed for oocytes undergoing prolonged culture after dissection (P < 0.05). Thus, zona dissection and sucrose exposure of freshly aspirated mature human oocytes do not result in classical oocyte activation.
The interaction between exogenous DNA and sperm cells
Authors: Lavitrano M. , French D. , Zani M. , Frati L. , Spadafora C.
Published: February 2005

Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15‐ to 20‐min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly‐L‐lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150–750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30–35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm–DNA interaction.
Evidence for the presence of an integrin cell adhesion receptor on the oolemma of unfertilized human oocytes
Authors: Fusi F. M. , Vignali M. , Busacca M. , Bronson Richard A.
Published: February 2005

The Arg‐Gly‐Asp peptide (RGD), contained in several extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen, is a tripeptide that plays a role as a recognition sequence in many cell‐to‐cell and cell‐to‐matrix adhesion mechanisms, through its interaction with several receptors of the integrin family. We previously described the ability of the oolemma of hamster oocytes to bind GRGDTP coupled to the surface of activated immunobeads and demonstrated that RGD‐containing oligopeptides inhibit the adhesion of human and hamster spermatozoa to zona‐free hamster oocytes and their subsequent penetration. In the present experiments, we show, utilizing immunobeads coated with an RGD‐containing peptide (PepTiteTM 2000), that the oolemma of unfertilized human eggs is also able to recognize this adhesion sequence. The binding of PepTiteTM 2000‐coated immunobeads to the oolemma was inhibited by the oligopeptide GRGDTP as well as by fibronectin and laminin. When immunobeads were prepared with a PepTiteTM concentration of 10 μg/ml, GRGDTP 150 μg/ml, laminin 80 μg/ml, and fibronectin 60 μg/ml inhibited bead rosetting on the egg surface. These data suggest that a specific binding moiety for RGD is present on the human egg surface. The binding of fibronectin to the oolemma was also demonstrated by the rosetting of immunobeads coupled with antifibronectin antibody to human oocytes after their exposure to 1 mg/ml free fibronectin. Such binding of fibronectin to the oolemma could be inhibited by coincubation with a monoclonal antibody directed against the cell adhesion fragment of fibronectin. In addition, oolemmal rosetting of immunobeads coupled with a monoclonal antibody directed against the α5 subunit, usually part of the fibronectin receptor VLA 5 (α5β1), provided additional evidence that a putative fibronectin receptor is present on the oolemma of human eggs.
Subzonal insemination with a single spermatozoon using manipulation assisted sperm adhesion onto the ooplasmic membrane in mouse ova
Authors: Kobayashi Kazuhiko , Okuyama Manabu , Fujimoto Goro , Ogawa Shyoso , Rothman Cappy M. , Hill David L.
Published: February 2005

A simple and successful method of microinjection of a single spermatozoon under the zona pellucida of a mouse oocyte has been developed. A characteristic of this method is that the tip of the sperm injection needle pierces the zona pellucida without touching the ooplasmic membrane. All the ova (277) used for this series of experiments had normal morphology after the injection procedure. Spermatozoa preincubated in culture medium for capacitation and those treated with ionophore A23187 for induction of acrosome reaction were used. In combination with some of these injections, a manipulation assisting the adhesion of the sperm head onto the ooplasmic membrane was employed. The fertilization rate (67.3%) of the ova injected with the ionophore‐treated sperm using the sperm‐adhesion treatment was significantly higher (P<0.005) than that obtained by the injection of the preincubated sperm without applying the adhesion treatment (23.6%). All three of the recipients that received the 24 fertilized ova became pregnant and gave birth to 11 off‐spring (45.8%). The inseminations performed with the sperm‐adhesion treatment using the immotile sperm from the preincubated population and/or those from the ionophore‐treated population did not result in fertilization in any case. These results suggest that the fertilization rate of subzonal insemination with motile ionophore‐treated sperm can be improved by applying the sperm‐adhesion treatment and that sperm motility might be involved in the establishment of fertilization, even after the adhesion of the sperm head with the mouse ovum membrane.
Germ cell‐specific expression of a proacrosin‐cat fusion gene in transgenic mouse testis
Authors: Nayernia Karim , Burkhardt Elke , Beimesche Stephan , Keime Sabine , Engel Wolfgang
Published: February 2005

Acrosin is a serine proteinase located in a zymogen form, proacrosin in the acrosome of the sperm. It is released as a consequence of the acrosome reaction and is believed to be the most important enzyme in the fertilization process. In the mouse, the proacrosin gene is transcribed premeiotically in spermatocytes, but protein biosynthesis starts in haploid spermatids and is restricted to the emerging acrosome. Four lines of transgenic mice harboring 2.3 kb of 5′ untranslated region of the rat proacrosin gene fused to the CAT‐reporter gene were generated by microinjection of fertilized eggs. The chimeric gene was found to be present in 10–100 copies per genome in the different strains. The 5′ untranslated region of rat proacrosin gene could properly direct CAT‐gene expression to spermatocytes and CAT‐mRNA translation to round spermatids as it is known for mouse proacrosin gene. However, CAT protein is not restricted to the acrosome; rather, it is distributed in the spermatid cytoplasm. This could be due to the lack of DNA sequences for a hydrophobic leader peptide that have been found in all mammalian proacrosins studied until now but that was not present in transgene. It can be concluded from our results that cis‐acting sequences required for tissue specific proacrosin expression reside on a 2.3‐kb restriction fragment and are conserved in the proacrosin genes of mouse and rat.
Localization of DNase I‐hypersensitive regions during rat spermatogenesis: Stage‐dependent patterns and unique sensitivity of elongating spermatids
Authors: McPherson S. M. , Longo F. J.
Published: February 2005

DNase I‐hypersensitivity of rat spermatogenic cells was analyzed (1) to establish overall patterns of hypersensitivity in individual cell types, (2) to correlate these patterns with known changes in chromatin organization and function, and (3) to provide a foundation for further analyses examining DNase I‐hypersensitivity and the localization of specific genes during spermatogenesis. Parameters for in situ nick translation, using radioactive and fluorescent probes to visualize DNase I‐hypersensitive regions (DHR), were established for fixed and sectioned testicular preparations, permeabilized cells, and isolated germ cell nuclei. As anticipated, the pattern of DHR changed in a cell‐type specific manner during the course of spermatogenesis, reflective of known stage‐dependent alterations in the composition and structure of both the chromatin and the nuclear lamina/matrix as well as changes in gene expression. DHR in preleptotene spermatocytes were primarily peripheral, while in pachytene spermatocytes they were localized along the condensed chromosomes. The pattern of DHR changed from “checkerboard” in steps 7–8 round spermatid nuclei to “lamellar” in steps 10–11 elongating spermatids. In steps 12–13 elongating spermatids, DHR were localized throughout the nuclei or in a graded manner—increasing from anterior to posterior and mirroring the pattern of chromatin condensation. However, unlike the case in other stages, DNA of steps 12–13 elongating spermatids was exquisitely sensitive to nick translation even in the absence of exogenous DNase I. In contrast to the labeling of earlier stages, steps 16–19 spermatids and mature spermatozoa did not demonstrate DNase I‐hypersensitivity under any conditions employed. A variety of agents that interact with topoisomerase II and DNA (teniposide, novobiocin, ethidium bromide, and adenosine triphosphate) were tested to determine the basis for the unique sensitivity to nick translation of steps 12–13 elongating spermatids. None of the agents tested, however, affected this unique labeling. The sensitivity of steps 12–13 elongating spermatids to nick translation in the absence of exogenous nuclease indicates the presence of endogenous nicks, which may relieve torsional stress and aid rearrangement as the chromatin is packaged into a form characteristic of the mature spermatozoon.
Activation of a G protein in mouse sperm by the zona pellucida, an egg‐associated extracellular matrix
Authors: Wilde Mary W. , Ward Cynthia R. , Kopf Gregory S.
Published: February 2005

Mammalian sperm possess a guanine nucleotide‐binding regulatory protein (G protein), with properties similar to Gi, that appears to be involved in the signal transduction pathway required for zona pellucida (ZP)‐mediated acrosomal exocytosis. Mouse sperm treated with pertussis toxin (PT), a toxin that functionally inactivates Gi proteins, bind to the ZP of mouse eggs but are inhibited from undergoing acrosomal exocytosis. We have measured high‐affinity GTPase activity and GTPγ[35S] binding in mouse sperm homogenates incubated in the absence and presence of ZP glycoproteins isolated from either ovulated eggs or from ovarian homogenates to determine whether this extracellular matrix can activate the sperm‐associated Gi protein. An increase in GTP hydrolysis (∼50% over basal activity) and GTPγ[35S] binding (∼25–60% over basal activity) is observed when sperm homogenates are incubated in the presence of solubilized ZP glycoproteins, and the increase in GTPase activity is dependent on the concentration of ZP added to the homogenates. Accompanying this increase is a reduction in the ability of PT to catalyze in vitro [32P]ADP‐ribosylation of a Mr = 41,000 sperm Gi protein, suggesting that the increase in GTPase activity and GTPγ[35S] binding is associated with the activation of a PT‐sensitive sperm G protein(s). The ability of the ZP to stimulate high‐affinity GTPase activity in these homogenates appears to be dependent on the capacitation state of the sperm from which the homogenates are prepared. These data suggest that a component(s) of the ZP may function in a manner similar to that of other ligands by binding to a sperm surface‐associated receptor and subsequently activating a G protein coupled to an intracellular signal transduction cascade(s) required for induction of acrosomal exocytosis.
Intracellular CA2+ response of rabbit oocytes to electrical stimulation
Authors: Fissore Rafael A. , Robl James M.
Published: February 2005

Electrical stimulation is known to cause activation in mammalian oocytes, possibly by eliciting an elevation in intracellular calcium (Ca2+). This study reports intracellular Ca2+ concentrations in mature rabbit oocytes using the Ca2+ indicator fura‐2. Calcium levels were determined prior to, during, and after the administration of an electrical pulse (3.6 kV/cm for 60 μsec). Baseline Ca2+ levels ranged from 30 to 90 nM. The intracellular Ca2+ transient evoked by a pulse, peaked at 11 sec, was highly variable in amplitude (40–300 nM) and returned to prepulse levels within 300 sec. Electrically stimulated oocytes did not exhibit repetitive Ca2+ transients. The size of the cytoplasmic Ca2+ rise was influenced by the duration of the pulse, the field strength and the concentrations of external Ca2+ (P < 0.05). Oocytes electrically stimulated in the presence of 100 μM CaCl2, which evoked Ca2+ transients with a mean magnitude of 120 nM, activated at a higher rate (P < 0.05) than oocytes stimulated in the presence of either higher or lower levels of external Ca2+. Although oocytes electrically shocked at 16–18 hr after administration of human chorionic gonadotropin (hphCG) activated at a lower rate than oocytes stimulated at 22–24 hphCG (P < 0.05), their intracellular Ca2+ response to the pulse was similar (P < 0.05). These results indicate that electrical pulse parameters and extracellular Ca2+ concentrations can be used to modulate intracellular Ca2+ levels and optimize oocyte activation rates. Furthermore, the data suggest that as the oocyte ages it becomes more responsive to a given intracellular Ca2+ elevation. © 1992 Wiley‐Liss, Inc.
Role of a germ cell‐specific sulfolipid‐immobilizing protein (SLIP1) in mouse in vivo fertilization
Authors: Tanphaichitr Nongnuj , Tayabali A. , Gradil C. , Juneja S. , Léveillé M. C. , Lingwood C. A.
Published: February 2005

Sulfolipid‐immobilizing protein 1 (SLIP1) is a germ cell plasma membrane protein that binds specifically to sulfogalactosylglycerolipid, a sulfoglycolipid found preferentially in mammalian male germ cells (Lingwood, Can. J. Biochem. Cell. Biol. 63:1077–1085, 1985b). SLIP1 in mouse and rat sperm exists on the periacrosomal membrane, where sperm initially bind to eggs. Using the in vitro mouse sperm‐egg binding assay with in vitro‐capacitated sperm, we obtained results previously suggesting that sperm SLIP1 is involved in mouse sperm‐zona pellucida interaction. In this study, using the in vitro sperm‐egg binding assay, we showed that SLIP1 in uterine sperm was similarly engaged in this process. Involvement of mouse sperm SLIP1 was also shown to be important in the vivo fertilization process. Superovulated females inseminated with caudal epidididymal and vas deferens sperm preexposed to anti‐SLIP1 lgG yielded only 20% fertilized zygotes, while 80% fertilization was observed in females inseminated with sperm preincubated with preimmune serum lgG. The lower fertilization rate was not due to changes in the sperm capacitation rate as assessed by chlortetracycline staining. © 1992 Wiley‐Liss, Inc.
Comparison of embryonic developmental competence of mouse oocytes grown with and without serum
Authors: Eppig John J. , Wigglesworth Karen , O'Brien Marilyn J.
Published: February 2005

The first objective of this study was to determine whether oocyte growth in serum‐free medium affects the solubility of the zona pellucida to α‐chymotrypsin digestion, which is an index of zona pellucida “hardening” and reflects the potential penetrability of the zona pellucida by sperm. Oocyte‐granulosa cell complexes were isolated from the preantral follicles of 12‐day‐old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum‐free medium. The zonae pellucidae of oocytes grown in serum‐free medium were four times as hard as freshly isolated germinal vesicle (GV)‐stage oocytes grown in vivo or oocytes grown in vitro in FBS‐containing medium. The hardening of the zonae pellucidae of oocytes grown in serum‐free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum‐free vs. FBS‐containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum‐free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two‐cell stage after insemination compared with only 6% of the ova grown in serum‐free medium. Addition of fetuin to the serum‐free medium used for oocyte growth increased the frequency of cleavage to the two‐cell stage. Of the embryos derived from oocytes that grew in FBS‐containing medium, 70% completed the two‐cell stage to blastocyst transition. In contrast, only about 40% (P < 0.01) of the embryos derived from oocytes that grew in serum‐free medium completed this transition regardless of whether the medium for oocyte growth was supplemented with fetuin. Nevertheless, 12% of the two‐ to four‐cell‐stage embryos derived from oocytes that grew and matured in serum‐free medium containing fetuin developed to live young after transfer to the oviducts of foster mothers. These results suggest that serum‐borne growth factors may function during oocyte growth and maturation to promote events leading to successful fertilization and embryogenesis.
A transient rise in intracellular Ca2+ is a precursor reaction to the zona pellucida‐induced acrosome reaction in mouse sperm and is blocked by the induced acrosome reaction inhibitor 3‐quinuclidinyl benzilate
Authors: Storey Bayard T. , Hourani Catherine L. , Kim J. B.
Published: February 2005

The acrosome reaction induced by the zona pellucida in mouse sperm has been shown to proceed in two stages experimentally distinguishable by the fluorescent probe chlortetracycline. Entry into the first stage of sperm bound to isolated, structurally intact zonae pellucidae is blocked by the compound 3‐quinuclidinyl benzilate. In this study, we show, utilizing the fluorescent Ca2+ indicator fluo‐3, that the first stage of the zona‐induced acrosome reaction is characterized by an increase in intracellular Ca2+, followed by a decrease as the acrosome reaction proceeds. This calcium transient is completely suppressed by 3‐quinuclidinyl benzilate. We conclude that the Ca2+ transient is induced by the zona pellucida and is required for the zona‐induced acrosome reaction. Blockage of this sperm intracellular Ca2+ transient provides a mechanism for the inhibitory action of 3‐quinuclidinyl benzilate on the zona‐induced acrosome reaction in mouse sperm. © 1992 Wiley‐Liss, Inc.
Specific distribution of an epididymal 50 KDa protein revealed by an anti‐mos protein monoclonal antibody
Authors: Koyama Yoh‐Ichi , Ishikawa K. , Marunouchi T.
Published: February 2005

An anti‐Mos protein monoclonal antibody, 4A6, was used to investigate the distribution of the antigen in the epididymis, in which the c‐mos gene is reportedly expressed. The 4A6‐reactive antigen was found on the basement membrane and luminal surface of the epithelial cells in the caput epididymis of BALB/c male mice as well as in the proximal corpus epididymis, the cauda epididymis, and the vas deferens. The 4A6 antigen was also found on the luminal surface of the epithelial cells in the epididymis of male germ cell‐deficient C57BL/6J‐Wv/Wv mice. This confirmed that the 4A6 antigen does not derive entirely from the testicular c‐Mos protein but is synthesized in the epididymis. Western blot analysis revealed that the molecular weight of the epididymal 4A6 antigen was 50 kDa, which is unusually high for the c‐Mos protein. With its specific distribution in the epididymis, the protein should play a specific role in functions of the epididymis. © 1992 Wiley‐Liss, Inc.
Clusterin (SGP‐2) in epididymal luminal fluid and its association with epididymal spermatozoa in androgen‐deprived rats
Authors: Mattmueller Danette R. , Hinton Barry T.
Published: February 2005

Clusterin is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells and epididymal epithelium. The goal of this study was to determine the presence of clusterin in the luminal fluid of the cauda epididymides and its association with the membranes of developing spermatozoa in the presence and absence of androgen. We have previously demonstrated by two‐dimensional (2‐D) Western blot probing for clusterin that in epididymal fluid the amounts of clusterin were: caput < corpus < cauda. Luminal fluid from cauda epididymides was collected from control and orchiectomized rats (6 and 12 days) and orchiectomized animals that received testosterone implants. Equal volumes of fluid were analyzed by 2‐D Western blot probing for clusterin. Following orchiectomy, there was an increase in clusterin in the luminal fluid after 6 days and maximal amount after 12 days compared with control cauda fluid. Orchiectomized animals which received testosterone treatment showed levels of clusterin comparable to that of controls. Serum clusterin was detected in fluid of orchiectomized animals with and without testosterone. Western blots of cauda sperm membrane extracts of control animals and orchiectomized animals treated with testosterone had a very low level of epididymal clusterin, whereas extracts collected from orchiectomized animals revealed high levels of clusterin. We suggest that, in the normal animal, clusterin is secreted into the lumen of the proximal epididymis where it binds to the sperm membrane. In the distal epididymis, clusterin dissociates from sperm and is processed (proteolysis/endocytosis). We hypothesize that, in the absence of androgen, the processing and regulation of clusterin is disrupted. © 1992 Wiley‐Liss, Inc.
Tight junctions and cavitation in the human pre‐embryo
Authors: Gualtieri Roberto , Santella Luigia , Dale Brian
Published: February 2005

In the human morula, tight junctions are found between all cell pairs, at all levels of cellular apposition, associated with underlying masses of microfilaments. In cavitating morula, lanthanum tracer gained access to the intercellular spaces, except at the intersections with nascent extracellular cavities, marking the first assembly of zonulae occludentes. Presumptive trophectoderm cells contained vacuoles and larger cavities often associated with secondary lysosome‐like bodies. Since the vacuoles and intracellular and extracellular cavities contain electron‐dense polygranules of about 23 nm diameter, they may have common origins. In trophectoderm cells of the early blastocyst, the large intracellular vacuoles and cavities were absent, and the zonulae occludentes were located apically. Mechanisms for nascent blastocoele formation are discussed. © 1992 Wiley‐Liss, Inc.
Modulation of vascular cell behavior by transforming growth factors β
Authors: Madri Joseph A. , Bell Leonard , Merwin June Rae
Published: February 2005

The vascular cell responses to the type 1, 2, and 3 isoforms of transforming growth factor‐β (TGF‐β1, TGF‐β2, TGF‐β3) were studied using bovine aortic endothelial (BAECs) and smooth muscle cells (BASMC3) as well as rat epididymal fat pad microvascular endothelia (RFCs). Three distinct bioassays indicated that TGF‐β3 elicits results that do not differ significantly from those of the TGF‐β1 isoform in all three cell populations. These assays are: inhibition of proliferation, cell migration, and neovascularization. By contrast the cellular responses to TGF‐β1 and TGF‐β3 differed from those to TGF‐β2. Three distinct receptor assays revealed the preesnce of type I and type II TGF‐β1 cell surface binding proteins on BAECs, BASMCs, and RFCs. Experimentation to decipher cell surface binding by the different isoforms revealed that iodinated TGF‐β1 bound to the surface of all three vascular cell types can be competed off in similar fashion by either TGF‐β1 or TGF‐β3; however, competition with TGF‐β2 produced unique binding profiles dependent on the cell type examined. The ratios of type I to type II TGF‐β receptors in these three vascular cell types vary from 1:1 in BAECs to 1.5:1 in RFCs to 3:1 in BASMCs and can be correlated with the differences noted in cellular responses to TGF‐β1 and TGF‐β2 in proliferation, migration, and in vitro angiogenic assays.

In summary, both the TGF‐β1 and TGF‐β3 isoforms of the transforming growth factor‐β family evoke comparable responses in proliferation, migration, angiogenic and cell surface bindinga ssays using three distinct vascular cell types, while the biofunctions of TGF‐β2 on these cells are distinct. These findings support the hypothesis that there are different responses to the TGF‐βs depending on the cell type and experimental conditions as well as the TGF‐β concentration and isoform. © 1992 Wiley‐Liss, Inc.
Müllerian inhibiting substance in reproduction and cancer
Authors: Donahoe Patricia K.
Published: February 2005

During embryogenesis normal male phenotypic development requires the action of Müllerian Inhibiting Substance (MIS) which is secreted by Sertoli cells of the fetal testis. As testes differentiate in genetic (XY) males, they produce MIS which causes regression of the Müllerian ducts, the anlagen of the female reproductive tract. Soon thereafter, testicular androgens stimulate the Wolffian ducts. In females, on the other hand, MIS is not produced by grandulosa cells until after birth, before which, estrogens induce Müllerian duct development, while the Wolffian ducts passively atrophy in the absence of androgenic stimulation. High serum MIS levels in males are maintained until puberty, whereupon they fall to baseline levels. In females MIS is undetectable in serum until the peripubertal period when values approach the baseline levels of males. This distinct pattern of sexual and ontogenic expression presupposes and requires tight regulation.

MIS may play a role in gonadal function and development. Our laboratory has shown that an important role for ovarian MIS is to inhibit oocyte meiosis, perhaps providing maximal oocyte maturation prior to selection for ovulation and subsequent fertilization. Furthermore, Vigier et al. (Development 100:43–55) have recently obtained evidence that MIS may influence testicular differentiation, coincident with inhibition of aromatase activity. Current structure‐function studies demonstrate that MIS, like other growth regulators in its protein family, requires proteolytic cleavage to exhibit full biological activity. MIS can be inhibited by epidermal growth factor. This antagonism, which is common to all MIS functions so far investigated, is associated with inhibition of EGF receptor autophosphorylation. We have provided evidence that bovine MIS can inhibit female reproductive tract tumors arising in adults. More recent work with highly purified recombinant human MIS (rhMIS) has extended these early observations, showing that this recombinantly made fetal inhibitor can also suppress tumors of Müllerian duct origin and thus may prove to be useful in the clinical management of gynecologic neoplasms. Recent investigations have identified ocular melanoma as another potential target for rhMIS therapy in humans. © 1992 Wiley‐Liss, Inc.
TGF‐β regulation of epithelial cell proliferation
Authors: Moses Harold L.
Published: February 2005

The predominant effect of TGF‐β1 on cell proliferation is inhibition. Earlier studies demonstrated that TGF‐β1 inhibition of skin keratinocyte proliferation involves suppression of c‐myc transcription and indirect evidence suggested that the protein product of the retinoblastoma gene (pRB) may be involved in this process. Skin keratinocytes transformed by SV40 and human papilloma virus‐16 (HPV‐16) or HPV‐18 resisted growth inhibition and suppression of c‐myc mRNA by TGF‐β. Transient expression of HPV‐16 E7 gene, adenovirus E1A, and SV40 large T antigen (TAg) blocked the TGF‐β1 suppression of c‐myc transcription. Studies with transformation‐defective mutants of E1A and TAg suggested that a cellular protein(s) that interacts with a conserved domain of the DNA tumor virus oncoproteins mediates TGF‐β1 suppression of c‐myc transcription and keratinocyte growth. Transient expression of pRB in skin keratinocytes repressed human c‐myc promoter/CAT transcription as effectively as TGF‐β1. The same c‐myc promoter region, termed the TGF‐β Control Element (TCE), was required for regulation by both TGF‐β1 and pRB. TCE bound a cellular protein of approximately 106 kDa and this binding was decreased by TGF‐β1 treatment. Our data indicate that pRB can inhibit c‐myc transcription and suggest the involvement of cellular factor(s) in addition to pRB in the TGF‐β1 pathway for the suppression of c‐myc transcription and growth inhibition. The possible involvement of pRB in the TGF‐β1 pathway for suppression of c‐myc transcription has a number of implications; c‐myc is a cellular proto‐oncogene involved in positively regulating cell proliferation. TGF‐β1 may therefore act through the tumor suppressor gene product, pRB, to negatively regulate c‐myc transcription and subsequently cell growth. This would implicate tumor suppressor genes in the response pathway for diffusible growth inhibitors, perhaps analogous to nuclear proto‐oncogene involvement in the growth factor pathway. © 1992 Wiley‐Liss, Inc.
Construction of the nuclear matrix at the transition from maternal to zygotic control of development in the mouse: An immunocytochemical study
Authors: Prather R. S. , Schatten G.
Published: February 2005

The nuclear matrix is thought to be responsible for DNA organization, DNA replication, RNA synthesis, and RNA processing. We have looked for the presence of nuclear matrix antigens during early mouse embryogenesis. Antibodies to peripheral and interior antigens (P1, PI1, PI2, and lamin B) were used to immunolocalize nuclear matrix antigens in germinal vesicle oocytes, metaphase II oocytes, zygotes, two‐cell‐stage embryos, and eight‐cell stage embryos. All antibodies reacted with the nuclei of germinal vesicle oocytes, and two‐ and eight‐cell‐stage embryos; however, only P1 and lamin B were present at the pronuclear stage. In eggs collected at the pronuclear stage and cultured to the late two‐cell stage in the presence of α‐amanitin, the matrix morphology was altered for PI1 and PI2. α‐Amanitin had no affect on the distribution of P1 or lamin B antigens. If α‐amanitin was added 2 hr after cleavage to the two‐cell stage, the normal staining pattern of PI2 was retained. These results suggest that the presence of specific components of an internal matrix is correlated with normal genomic activity.
Multiple sites of HOX‐7 expression during mouse embryogenesis: Comparison with retinoic acid receptor mRNA localization
Authors: Lyons Gary E. , Houzelstein Denis , Sassoon David , Robert Benoîct , Buckingham Margaret E.
Published: February 2005

We report results from a study of Hox‐7 expression during mouse embryonic and fetal development and compare the localization of Hox‐7 transcripts with those of the retinoic acid receptors. Transcripts were detected by in situ hybridization. Hox‐7 expression occurs in (1) cephalic neural crest and its derivatives, (2) sites of ectomesodermal interaction, (3) extraembryonic tissues, and (4) endocardial cells. Hox‐7 does not seem to be involved in defining rostrocaudal boundaries, but instead appears to be expressed along the proximodistal axes at these sites. We further investigated the active sites of morphogenesis, which involve an ectomesodermal interaction (e.g., limb buds, visceral arches), including genital tubercle and tail ridge. These are regions highly positive for Hox‐7 transcripts, and many are known to be sites for the expression of γ‐retinoic acid receptors (RARs) and cellular retinoic acid binding proteins. Most regions that express Hox‐7 are subregions of γ‐RAR expression. In the developing limb bud, expression of Hox‐7 takes place in the interdigital region, where it overlaps areas of β‐RAR expression.
Regulated temporal and spatial expression of the calcium‐binding proteins calcyclin and opn (osteopontin) in mouse tissues during pregnancy
Authors: Waterhouse Paul , Parhar Ranjit S. , Guo Xiaojia , Lala Peeyush K. , Denhardt David T.
Published: February 2005

In situ hybridization and northern/slot blot analyses were used to quantify the expression of calcyclin (2A9, 5B10), osteopontin (opn, secreted phosphoprotein, 2ar) and calmodulin mRNAs in mouse tissues that support pregnancy. High‐to‐moderate levels of the mRNAs of all three genes were detected at discrete locations in the uterus, decidua and placenta as a function of gestation time. Calmodulin expression was constant in these tissues; calcyclin mRNA was high during early pregnancy and declined after day 8–9 of gestation; and opn mRNA was undetectable before day 7, with maximal levels on days 9–12 in each of these tissues. Calcyclin, but not opn, expression was also observed in the chorioamnion after day 12. Calcyclin was expressed throughout the decidua on day 8 but became restricted to the primary (antimesometrial) decidual zone and decidua lateralis on day 9, and the decidua capsularis after day 9. By contrast, opn mRNA was localized on day 9 to the mesometrial triangle, which contains a large population of granulated metrial gland cells, and to the decidua basalis. These two genes may serve as markers for the two types of decidual tissue. We suggest that one function of OPN, which may be an indicator of cells in the decidua that have a bone marrow genealogy, is to mediate the flux of calcium from the maternal circulation to the developing embryo.
Expression of interleukin‐6 in porcine, ovine, and bovine preimplantation conceptuses
Authors: Mathialagan Nagappan , Bixby James A. , Roberts R. Michael
Published: February 2005

A porcine interleukin‐6 (pIL6) cDNA has been cloned from pig spleen cDNA library to provide information that would allow us to study IL‐6 mRNA expression during pregnancy of several domestic Artiodactyla. The cDNA is 1058 bp long and with a single open reading frame that encodes a 212 amino acid polypeptide with 28‐residue signal sequence. It shares 61% and 43% amino acid sequence identity with human and mouse IL‐6, respectively. PCR procedures with primers designed from regions of sequence conserved between human and pig have been used to identify IL‐6 cDNA in λgt11 libraries constructed from day 15–16 (sheep), day 17 (cattle), and day 13–17 (pig) conceptus mRNA. The presence of IL‐6 mRNA in elongating preimplantation ovine (days 13–25), porcine (days 13–21), and bovine (days 16–20) conceptuses was also demonstrated by PCR after reverse transcription of total ribonucleic acid with reverse transcriptase and by solution hybridization with a pIL‐6 cRNA probe. These observations suggest that IL‐6 is a product of these early conceptuses and may be involved in early maternal responses to the presence of an embryo within the uterus.
Changes in camp phosphodiesterase activity and cAMP concentration during mouse preimplantation development
Authors: Dardik Alan , Schultz Richard M.
Published: February 2005

Cyclic nucleotide phosphodiesterase (PDE) activity and cAMP amounts were measured in mouse preimplantation embryos at the 1‐cell, 2‐cell, 8‐cell/ morula, and mid‐blastocyst stages. PDE activity remained constant between the 1‐cell and 2‐cell stages. It decreased by the 8‐cell stage and continued to decrease by the mid blastocyst stage to about 14% of the 1‐and 2‐cell values. By contrast, cAMP amounts remained essentially constant at 0.05 fmole/embryo (0.3 μM) from the 1‐cell to the blastocyst stage and increased to 0.175 fmole in the fully expanded blastocyst that was close to hatching. Measurements of embryo volume indicated that intracellular volume remained essentially constant up to the blastocyst stage. The morphological changes in cell shape that accompany differentiation of the trophectoderm and that are coupled with blastocoel expansion decresed the intracellular volume. This decrease resulted in an increase in the cAMP concentration to about 0.4 μM by the mid‐blastocyst stage. Previous studies indicate that either cAMP or TGF‐α/EGF can stimulate the rate of blastocoel expansion. Although TGF‐α/EGF can elevate cAMP levels in other cell types, TGF‐α, at a concentration that maximally stimulates the rate of blastocoel expansion, did not elevate cAMP in blastocysts. Thus, it was unlikely that elevation of cAMP is the mechanism by which TGF‐α stimulates the rate of blastocoel expansion.
Migration of centromere proteins in rabbit spermatids
Authors: Courtens Jean‐Luc , Biggiogera Marco , Rothfield Naomi F. , Burnier Michel , Fakan Stanislav
Published: February 2005

Human autoantibodies were used to localize centromere proteins by immunoelectron microscopy, immunofluorescence, and confocal microscopy in isolated cells and in cryosections of rabbit testis. A computer‐assisted three‐dimensional reconstruction of the positions and sizes of fluorescent spots allowed us to follow their movements during the different phases of spermiogenesis. In very young spermatids, the centromeres were distributed within a space separated from both the external nuclear limits and the nuclear core. They moved towards the nuclear limits and the nuclear core. They moved towards the nuclear center in cap phase spermatids, where they clustered into a few large centromeric masses. In preelcngating spermatids, the immunolabeled proteins were dispersed within an equatorial area, where they formed one large mass. In late spermatids, the mass moved towards the posterior part of the nucleus, and, in the spermatozoon, the two basal knobs located at the base of the nuclei were the only strongly immunolabeled structures, while no labeling of the main part of the nucleus was observed. Since the number of centromeres remained close to the number of chromosomes until the cap phase of spermatid differentiation, we hypothesize that the labeling of young spermatids corresponds to centromeric proteins associated with their specific DNA counterparts, while the centromere proteins, possibly detached from their DNA loci, were released from nuclei of old spermatids in the same way as are histones and transition proteins.
The fall of biological maturation promoting factor (MPF) and histone H1 kinase activity during anaphase and telophase in mouse oocytes
Authors: Fulka R. J. , Jung T. , Moor R. M.
Published: February 2005

Cell fusions have been used to determine the biological activity of the MPF complex in murine oocytes during their progression through anaphase and telophase to metaphase II. Oocytes (1) at metaphase I, (2) during the anaphase‐telophase transition, or (3) at metaphase II were fused to germinal vesicle‐staged (immature) oocytes. The hybrids were cultured for 1 h in the presence of db cAMP before fixation and nuclear evaluation. Metaphase! oocytes invariably induced germinal vesicle breakdown (GVBD) in the immature partner. By contrast, anaphase/telophase oocytes never induced GVBD in immature oocytes. The capacity to induce GVBD reappears after the formation of the second metaphase plate. In a second study, histone H1 kinase activity was measured during mouse oocyte maturation in single oocytes. H1 kinase activity was low in GV oocytes, increased sharply at MI, declined during anaphase and telophase and increased again at MII. After egg activation, H1 kinase activity was reduced to basal levels. These results provide direct evidence that a drop in activity of MPF in murine oocytes occurs concomitantly with the exit from metaphase I; MPF activity remains low until the cell re‐enters metaphase.
Collection of acrosome‐reacted human sperm using monoclonal antibody‐coated paramagnetic beads
Authors: Okabe Masaru , Matzno Sumio , Nagira Morio , Ying Xing , Kohama Yasuhiro , Mimura Tsutomu
Published: February 2005

A monoclonal antibody (MAb) against human acrosome‐reacted sperm was attached to paramagnetic polystyrene beads. Human sperm prepared by the swim‐up method were (1) incubated in m‐BWW, (2) incubated and ionophore treated, or (3) incubated with 5% seminal fluid. After treatment, sperm were mixed with the beads and incubated for 1 hr. Variously treated sperm showed different binding abilities to the beads. Sperm bound to the beads were collected by a magnet and subjected to triple staining. Most of the collected sperm were acrosome reacted. The results suggested that the beads can be used to estimate the acrosomal status of sperm, and that the use of antibody‐coated paramagnetic beads provides a convenient way of collecting acrosome‐reacted sperm. The acrosomal status detected by the beads was also compared with the ability of sperm to fuse with zonafree hamster eggs. It was found that greater bead‐binding ability correlated with more sperm fusing with zona‐free hamster eggs.
Evidence for differential maturation of reciprocal sperm segregants in the murine RB(6.16) translocation heterozygote
Authors: Aranha Ivar P. , Martin‐DeLeon Patricia A.
Published: February 2005

The fertilizing ability of unaged sperm and those aged experimentally in the cauda by surgically ligating the corpus epididymis in males carrying the Rb(6.16) translocation was studied. Chromosomally normal females were inseminated with unaged sperm delivered by males mating at 3‐day intervals, and aged sperm were studied after matings on 6–14 postoperative days. The sperm chromosome complement was analyzed in first‐cleavage metaphase zygotes after sequential G‐ and C‐banding of the chromosomes. Of 283 metaphasic zygotes in the control group, 183 (or 64.7%) were analyzed and showed a ratio of 2.7:1 for chromosomally normal and balanced segregants of the translocation, deviating significantly (P < 0.001) from the expected 1:1. The ratio of X‐ to Y‐bearing sperm also deviated from expected (P > 0.01) mostly due to a significant deficiency (P > 0.05) of balanced sperm that were X‐bearing. Fertilized oocytes were recovered from matings of 10 males on days 6–8 post‐operatively, and, of 139 metaphasic one‐cell zygotes, 101 (or 72.3%) were analyzed. These showed a Mendelian ratio of 1:1 for normal and balanced segregants. The sex ration in the aged group (57Y:41X) also showed no deviation from 1:1. The results, which reveal significant physiological distortions for both the segregation and the sex ratios in males heterozygous for the Rb(6.16) translocation, suggest that differential maturation of the translocation‐bearing sperm and the chromosomally normal reciprocal exists. The findings further support the concept that sperm chromosomal complement affects their maturation and function, and that factors on chromosome 6 and the X or Y chromosome additively affect sperm function. © 1992 Wiley‐Liss, Inc.