bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphology and physiological significance of parathyroid glands in reptilia
Authors: Srivastav Ajai Kumar , Sasayama Yuichi , Suzuki Nobuo
Published: February 2005

Adult reptiles possess one or two pairs of parathyroid glands that have been shown in many species to derive from the third and fourth pharyngeal pouches, respectively. Up to five pairs may develop during early embryonic life. Excess glands may involute during late embryogenesis. The location of the parathyroid glands differs in the various species. As a general rule, they lie just anterior to the heart, the anterior pair (parathyroid III) being associated with the carotid artery, the posterior pair (parathyroid IV) with the aortic arch. In snakes, however, the anterior pair (parathyroid III) is associated with the carotid artery near the angle of the jaw. As shown by light microscopy and, to a lesser extent, by electron microscopy, the parathyroid parenchyma comprises secretory cells which may form dark and light variants, occasional oxyphil cells, and stellate cells. They are arrangend in cords separated by connective tissue containing a capillary network. Parathyroid secretory cells often form follicles which might be the result of degeneration. Degeneration may occur as a form of involution during winter in species undergoing seasonal changes. The product of parathyroid cells, the parathyroid hormone, is responsible for the maintenance of blood calcium concentration. The sites of action—bones, kidneys, intestine, endolymphatics, and dermal skeleton—are not well understood or not investigated. In some turtles, parathyroid hormone is not the (main) factor for the regulation of calcium homeostasis. © 1995 Wiley‐Liss, Inc.
Blood vascular bed and pericapillary space in rat parathyroid glands
Authors: Murakami Takuro , Tanaka Toshihisa , Taguchi Takehito , Ohtsuka Aiji , Kikuta Akio
Published: February 2005

The blood vascular bed and pericapillary space of the rat parathyroid gland were studied by scanning electron microscopy of vascular casts, freeze‐cracked tissue blocks, and NaOH‐treated tissue specimens. The findings were supplemented by transmission light and electron microscopy of sectioned tissue samples. The rat parathyroid gland contained a rich network of freely anastomosing capillaries. These capillaries were surrounded by marked pericapillary spaces that were demarcated by basal lamina of both capillaries and parenchymal cells. The pericapillary spaces contained many collagen fibrils and frequently issued some projections running deep into the sheets of parathyroid cells. The latter projections may be useful to supply the parenchymal cells located far from the capillaries. The collagen fibrils may regulate the flow of tissue fluid in the pericapillary space and convey parathyroid hormone, which is released at the apicolateral domain, into the capillaries. © 1995 Wiley‐Liss, Inc.
The biological significance of storage granules in rat parathyroid cells
Authors: Setoguti T. , Inoue Y. , Wild P.
Published: February 2005

Both prosecretory and storage granules are concomitantly formed at the trans Golgi network including the innermost Golgi cisterna. Prosecretory granules develop into small secretory granules that release their contents by exocytosis finely regulated by a complex mechanism for maintaining calcium homeostasis. In the rat parathyroid cells, storage granules are large secretory granules storing parathyroid hormone for an emergency supply. The hormone is rapidly discharged by exocytosis when serum calcium concentration is decreased. The granules are constantly produced even under conditions of low serum calcium concentration in the regions of 8 mg/dl. The granule content is constantly hydrolyzed when not discharged, leading to a decreased core and finally to the formation of vacuolar bodies. The fate of the vacuolar bodies is unknown. Hypercalcemic conditions accelerate hydrolysis. The threshold value of calcium concentration required for the release of storage granule contents is between 8.0 and 7.5 mg/dl and that of calcium concentration for accelerating degradation of storage granules is about 11.5 mg/dl. Sympathetic stimulation causes storage granules to be discharged regardless of hypercalcemia or hypocalcemia. Parasympathetic stimulation accelerates hydrolysis. The degradation of storage granules seems to be closely associated with an intracellular regulatory mechanism for parathyroid hormone secretion. © 1995 Wiley‐Liss, Inc.
Murine model of genetic demyelinating disease: The twitcher mouse
Authors: Suzuki Kinuko , Taniike Masako
Published: February 2005

Twitcher mouse is an authentic murine model of human genetic demyelinating disease, globoid cell leukodystrophy (GLD), or Krabbe disease. Since its discovery at the Jackson Laboratory (Bar Harbor, ME) this model has been used extensively for the morphological, biochemical‐enzymatic studies to clarify pathogenesis and also for therapeutic manipulation of genetic demyelinating disease in humans. As a result of these studies, now we know that (1) GLD is caused by a deficiency of lysosomal enzyme galactosylceramidase, and a toxic metabolite, psychosine, accumulates in the tissue, including the nervous system, damaging myelin forming cells and resulting in secondary demyelination; (2) morphological features of demyelination and associated cellular reactions in demyelination in this mutant are similar to those seen in autoimmune or toxic demyelination; and (3) with enzyme supplementation provided by bone marrow transplantation, remyelination occurs to some extent in demyelinated fibers in both central and peripheral nervous systems of twitcher mouse. © 1995 Wiley‐Liss, Inc.
Comparison of different drying procedures for scanning electron microscopy using human leukocytes
Authors: Ting‐Beall H. Ping , Zhelev Doncho V. , Hochmuth Robert M.
Published: February 2005

Using human leukocytes as test specimens, three different drying procedures for scanning electron microscopy: critical‐point drying (CPD), Peldri II, and tetramethylsilane (TMS), were compared. All three procedures produced identical surface morphology preservation. An equal amount of volume shrinkage was observed regardless of the dehydrants and drying techniques employed. Considering the simplicity, convenience, and time saved, air‐drying with TMS is by far the best choice for preparing animal cells for scanning electron microscopy. © 1995 Wiley‐Liss, Inc.
Macro, micro, and molecular research on spermatogenesis: The quest to understand its control
Authors: Kerr J. B.
Published: February 2005

Synchronous maturation of the germ cells in the seminiferous epithelium has long been recognized by microscopy, and is believed to be a consequence of a complex interaction between the germ cells and the Sertoli cells, largely driven by testosterone and its synergistic action with follicle‐stimulating hormone. Overall coordination of the cycle of the seminiferous epithelium is reviewed with regard to the known and possible actions of testosterone upon the Sertoli cells and the germ cells. With gradual refinements of optical instrumentation and development of a wide range of histological, morphometric, biochemical, and molecular techniques, coupled with selective alterations of hormonal stimulation and the cellular composition of the testis, new approaches to the question of how sperm production is regulated are becoming available. Germ cell and Sertoli cell functions are intimately related to each other via local, intratesticular, or paracrine signals which are suppressed or triggered at certain defined steps in the spermatogenic process. The coordination of germ cell proliferation and maturation is discussed in terms of the contributions made by microscopical techniques. © 1995 Wiley‐Liss, Inc.
Efficiency of spermatogenesis
Authors: Johnson Larry
Published: February 2005

Spermatogenesis is a process of division and differentiation by which spermatozoa are produced in seminiferous tubules. A measure of efficiency of spermatogenesis is the estimated number of spermatozoa produced per day per gram of testicular parenchyma. This measure is not influenced by species differences in testicular size; however, it is influenced by species differences in the numerical density of germ cells and in the life spans of these cells. Seminiferous tubules are composed of somatic cells (myoid cells and Sertoli cells), and germ cells (spermatogonia, spermatocytes, and spermatids). Activity of these three germ cells divide spermatogenesis into spermatocytogenesis, meiosis, and spermiogenesis, respectively. Spermatocytogenesis involves mitotic cell division to increase the yield of spermatogenesis and to produce stem cells and primary spermatocytes. Meiosis involves duplication and exchange of genetic material and two cell divisions that reduce the chromosome number and yield four spermatids. Spermiogenesis is the differentiation of spherical spermatids into mature spermatids which are released at the luminal free surface as spermatozoa. The spermatogenic Cycle is superimposed on the three major divisions of spermatogenesis. Spermatogenesis and germ cell degeneration can be quantified from numbers of germ cells in various steps of development throughout spermatogenesis, and quantitative measures are related to number of spermatozoa in the ejaculate. Germ cell degeneration occurs throughout spermatogenesis; however, the greatest impact occurs during spermatocytogenesis and meiosis. Efficiency of spermatogenesis is related to the amount of germ cell degeneration, pubertal development, season of the year, and aging of humans and animals. Number of Sertoli cells and amount of smooth endoplasmic reticulum of Leydig cells (but not Leydig cell number) are related to efficiency of spermatogenesis. In humans, efficiency of spermatogenesis is reflected in number of spermatogenic stages per cross‐section and number of missing generations within each stage; however, the arrangement of stages along the tubular length does not reflect differences in the efficiency of spermatogenesis. In short, spermatogenesis involves both mitotic and meiotic cell divisions and an unsurpassed example of cell differentiation in the production of the spermatozoon, and daily sperm production per g parenchyma is a measure of its efficiency. © 1995 Wiley‐Liss, Inc.
Distribution of Sertoli cell microtubules, microtubule‐dependent motors, and the Golgi apparatus before and after tight junction formation in developing rat testis
Authors: Redenbach Darlene M. , Hall Eric S. , Boekelheide Kim
Published: February 2005

Sertoli cells are polarized epithelial cells of the seminiferous epithelium which provide structural and physiological support for differentiating germ cells. They establish different basal and adluminal environments for the selective nurturing of pre‐ and post‐meiotic germ cells within the seminiferous epithelium, segregated by the Sertoli‐Sertoli cell tight junctional complex, the blood‐testis barrier. Tight junction formation between epithelial cells in vitro is a critical polarizing event associated with changes in polarized targeting of membrane‐specific proteins and reorganization of microtubules, centrioles, and the Golgi apparatus. To investigate whether tight junction formation is associated with organelle reorganization in Sertoli cells in vivo, we have characterized distribution patterns of Sertoli cell microtubules, the mechanoenzymes kinesin and cytoplasmic dynein, and the Golgi apparatus during tight junction formation in developing rat testis. Immunocytochemistry on samples taken at 5, 10, 15, 20, and 25 days of age was used to examine the distribution of these proteins during the extensive cellular reorganization that culminates in the formation of the blood‐testis barrier at 19 days of age. Our data show that the distribution patterns reflect the extensive intercellular repositioning of tubule cells in developing seminiferous tubules, but that changes in intracellular organization are not temporally associated with formation of the blood‐testis barrier. © 1995 Wiley‐Liss, Inc.
Novel Experimental Technique for the Determination of Monomer Droplet Size Distribution in Miniemulsion
Authors: do Amaral Marcelo , Bogner Agnès , Gauthier Catherine , Thollet Gilbert , Jouneau Pierre‐Henri , Cavaillé Jean‐Yves , Asua José M.
Published: February 2005

Summary: The lack of accurate knowledge for measuring monomer droplet size and droplet size distribution has hampered the further progress of miniemulsion polymerization. Monomer droplet size is probably the most important characteristics of a miniemulsion, influencing the miniemulsion stability and the nucleation mechanism. To date, several experimental techniques have been tested to measure miniemulsion droplet size, but none are convenient and accurate. This work presents a novel experimental technique, using a powerful new scanning transmission electron microscopy (STEM) imaging system, which allows transmission observations of wet samples in an environmental scanning electron microscopy (ESEM). This new imaging technique is a useful technique to directly measure droplet size and droplet size distribution.

Miniemulsion droplets in dark field wet STEM imaging conditions.

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Miniemulsion droplets in dark field wet STEM imaging conditions.
Diameter‐Dependence of the Morphology of PS‐b‐PMMA Nanorods Confined Within Ordered Porous Alumina Templates
Authors: Sun Yiming , Steinhart Martin , Zschech Danilo , Adhikari Rameshwar , Michler Goerg H. , Gösele Ulrich
Published: February 2005

Summary: Polystyrene‐block‐poly(methyl methacrylate) nanorods were prepared by wetting ordered porous alumina templates. We systematically investigated the diameter‐dependence of their morphologies by varying the pore diameters of the templates from 400 nm down to 25 nm. If the pore diameter exceeds the period of the block copolymer, the pores accommodate a non‐integer number of repeat periods. In case of smaller pores the occurrence of an ordered state could not be unambiguously verified.

TEM image of an ultra‐thin slice containing a cross‐section of a polystyrene‐block‐poly(methyl methacrylate) nanorod embedded in epoxy resin.

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TEM image of an ultra‐thin slice containing a cross‐section of a polystyrene‐block‐poly(methyl methacrylate) nanorod embedded in epoxy resin.
Long‐term efficacy and safety of pramipexole in advanced Parkinson's disease: Results from a European multicenter trial
Authors: Möller J. Carsten , Oertel Wolfgang H. , Köster Jürgen , Pezzoli Gianni , Provinciali Leandro
Published: February 2005

A double‐blind, placebo‐controlled study with a subsequent open‐label phase was conducted in 354 patients with Parkinson's disease (PD) and motor fluctuations under individually adjusted therapy with levodopa. During the double‐blind phase 174 patients received pramipexole and 180 placebo. In agreement with previous studies, pramipexole treatment improved UPDRS sum scores of parts II and III by 30% and off times by approximately 2.5 hours per day. Differences between the treatment groups became significant at a daily dose of 0.75 mg of pramipexole dihydrochloride. We, furthermore, performed post hoc analyses with respect to resting tremor and depression. Patients with pronounced resting tremor derived a clear benefit from pramipexole treatment compared with placebo. In addition, pramipexole significantly improved the subitems motivation/initiative and depression in a subpopulation with increased Unified Parkinson's Disease Rating Scale I scores at the time of inclusion. There were 262 patients who were subsequently enrolled into the open‐label study featuring a maximum duration of up to 57 months. Statistical analysis revealed good long‐term efficacy and tolerability of pramipexole. Overall, only a low prevalence of somnolence was found. In summary, this study provides additional level I evidence of the usefulness of pramipexole, suggests a particular tremorlytic and a possible antidepressant action of this compound, and addresses for the first time its efficacy and safety during long‐term administration in advanced PD. © 2005 Movement Disorder Society
Position‐dependent levitation of the dominant arm after left parietal stroke: An unreported feature of posterior alien limb syndrome?
Authors: Gondim F.A.A. , Oliveira G.R. , Cruz‐Flores S.
Published: February 2005

We describe a patient with an unreported feature of posterior alien limb phenomenon characterized by position‐dependent levitation of the dominant arm exacerbated by tactile stimulation and associated with low‐amplitude tremor of the fingers of the right hand in addition to a sensation of strangeness in the arm, secondary to a left parietal stroke. © 2005 Movement Disorder Society
A study by in situ hybridization of the stage of appearance and disappearance of the transition protein 2 and the mitochondrial capsule seleno‐protein mrnas during spermatogenesis in the mouse
Authors: Shih D. M. , Kleene K. C.
Published: February 2005

Transition protein 2 is a basic chromosomal protein which functions as an intermediate in the replacement of histones by protamines, and the mitochondrial capsule seleno‐protein is a constituent of the outer membrane of mitochondria which functions in constructing the mitochondrial sheath surrounding the flagellum. To determine precisely the stages in spermatogenesis when these mRNAs are present, paraffin sections of sexually mature testes were hybridized to 35S‐ and 3H‐labeled antisense RNAs and exposed to autoradiographic emulsion. The cell types hybridizing to probes in situ were determined by staining with hematoxylin and periodic acid Schiff. The in situ hybridizations reveal that the transition protein 2 mRNA is first detectable in step 7 round spermatids, persists at high levels through step 13, and is degraded before step 14. By contrast, the mitochondrial capsule seleno‐protein mRNA is first detected in step 3 round spermatids and persists at high levels until step 16, the end of spermiogenesis. The mitochondrial capsule seleno‐protein mRNA appears to be expressed only in haploid cells since low levels could not be detected in Northern blots of RNA from pachytene primary spermatocytes from 18 day prepubertal mice. These results demonstrate that the transition protein 2 and mitochondrial capsule seleno‐protein mRNAs are transcribed and degraded at different times during the haploid phase of spermatogenesis. © 1992 Wiley‐Liss, Inc.
Acrogranin, an acrosomal cysteine‐rich glycoprotein, is the precursor of the growth‐modulating peptides, granulins, and epithelins, and is expressed in somatic as well as male germ cells
Authors: Baba Tadashi , Hoff Henry B. , Nemoto Hiromi , Lee Hang , Orth Joanne , Arai Yuji , Gerton George L.
Published: February 2005

Spermatogenesis is a unique system of differentiation involving cellular remodeling and the biogenesis of sperm‐specific organelles. To study the biogenesis of one such organelle, the acrosome, we have been examining the gene expression, biosynthesis, and targeting of specific acrosomal proteins during mammalian spermatogenesis. An acrosomal marker that we recently purified and began characterizing is acrogranin, a 67,000‐molecular‐weight glycoprotein originally isolated from guinea pig testes. This glycoprotein is detected in pachytene spermatocytes and is found later in the acrosomes of developing spermatids and sperm. Immunoblotting of several tissues and immunofluorescent localization in frozen sections of guinea pig testes suggested that acrogranin was a germ cell‐specific glycoprotein that was expressed meiotically and post‐meiotically. However, Northern blot analysis demonstrated that the mRNA for acrogranin was ubiquitously expressed in all guinea pig and mouse tissues examined. Furthermore, the primary structures of guinea pig and mouse acrogranins, deduced from the cDNA sequences, reveal that this glycoprotein is a cysteine‐rich molecule with a motif that is tandemly repeated seven times, very similar to that of the human epithelin/granulin precursor. We conclude that guinea pig and mouse acrogranins are homologues of the precursor of the human and rat epithelin/granulin peptides previously demonstrated to have growth‐modulating properties. © 1993 Wiley‐Liss, Inc.
Human sperm–egg binding is inhibited by peptides corresponding to core region of an acrosomal serine protease inhibitor
Authors: Moore Alison , Penfold Linda M. , Johnson Jacqui L. , Latchman David S. , Moore Harry D. M.
Published: February 2005

An antiserum, designated R4 and raised against denatured hamster acrosomes, was shown to localize specifically to the acrosomal region of hamster, rat, mouse, and human spermatozoa, and to inhibit both hamster and human sperm–oocyte binding in vitro. Following screening of a human testis λgt11 cDNA expression library with the antiserum R4, a series of cDNA clones were isolated. One (cDNA 134) was selected based on the ability of the β‐galactosidase fusion protein to inhibit human and hamster sperm–zona binding in vitro. The fusion protein was also shown to inhibit the penetration of zona‐free hamster oocytes by human spermatozoa. Sequence analysis revealed that cDNA 134 coded for a portion of a serine protease inhibitor (serpin) closely related to plasma Protein C inhibitor. Sequencing of an additional cDNA clone (261) and Northern blot analysis confirmed that a Protein C inhibitor‐like mRNA is synthesised in the human testis. Affinity‐purified anti‐134 antibody specifically localized to the acrosomal region of both hamster and human sperm. Synthetic peptides corresponding to the conserved core region responsible for the interaction of the serpin with its cognate protease also blocked human sperm‐‐zona binding in vitro. The results suggest that this acrosomally located inhibitor plays an important role in the series of binding events that results in human fertilization. © 1993 Wiley‐Liss, Inc.
The expression of the H‐19 and IGF‐2 genes during human embryogenesis and placental development
Authors: Goshen Ran , Rachmilewitz Jacob , Schneider Tamar , de‐Groot Nathan , Ariel Ilana , Palti Zvi , Hochberg Abraham A.
Published: February 2005

The H‐19 gene in mice is maternally imprinted and its ectopic expression causes prenatal lethality. We have recently identified H‐19 transcript in differentiating human placental cells and showed that its expression increases concomitantly with differentiation of cytotrophoblasts in vitro. Placental and embryonal specimens were collected from conception products derived from normal first and second trimester pregnancy terminations. We investigated the abundance of H‐19 mRNA throughout placental development in vivo and compared it to the expression of other genes linked to placental differentiation. Furthermore, the expression of H‐19 transcript in different organs of human fetuses, aborted during the second trimester, was examined by RNA isolation from separated fetal organs. Since IGF‐2 is known to play an important role in embryogenesis, identical blots were hybridized with IGF‐2 probe. H‐19 expression in human placenta from the different trimesters of pregnancy remains practically constant. A high amount of H‐19 gene product was found in the fetoplacental unit with the highest level measured in the adrenal gland. These findings argue that H‐19 gene may play a role in human embryogenesis. © 1993 Wiley‐Liss, Inc.
Incorporation of [3H]inositol into phosphoinositides and inositol phosphates by rabbit blastocysts
Authors: Fahy Mary M. , Kane Michael T.
Published: February 2005

Preimplantation rabbit embryos collected at the early morula stage were cultured to blastocysts in the presence of [3H]inositol. The blastocysts were lysed, and both the aqueous and lipid portions were analysed for incorporated radioactivity. Thin‐layer chromatographic separation of the lipid portion indicated that [3H]inositol was incorporated into phosphatidylinositol, phosphatidylinositol 4‐phosphate, and phosphatidylinositol 4,5‐bisphosphate. HPLC anion‐exchange chromatography indicated that [3H]inositol was incorporated into inositol phosphates, including the two second messengers, inositol 1,4,5‐trisphosphate and inositol 1,3,4,5‐tetrakisphosphate, and also inositol monophosphate and inositol 1,4‐bisphosphate. These results provide evidence that rabbit blastocysts may have an active phosphatidylinositol second messenger system, which may be responsive to intrauterine factors or intraembryonic paracrine factors. © 1993 Wiley‐Liss, Inc.
Differences in regulation of testis specific lactate dehydrogenase in rat and mouse occur at multiple levels
Authors: Salehi‐Ashtiani Kourosh , Goldberg Erwin
Published: February 2005

The testis specific form of lactate dehydrogenase (LDH‐C4) is encoded by a single locus, Ldh‐c, and is tightly regulated in a tissue specific manner. Here we show differences in expression of Ldh‐c between rat and mouse, and describe the levels at which regulation of this gene differs in the two species. Our results demonstrate that the Ldh‐c message level is nearly nine fold greater in mouse testis and remains high post‐meiotically. In contrast, rat Ldh‐c mRNA is highest in primary spermatocytes and reduced in spermatids. The results of nuclear run‐on assays indicate that the transcription rate of Ldh‐c is only moderately higher in mouse than rat, and cannot account for a significant portion of the observed differences. Similar decay rates for both rat and mouse Ldh‐c mRNA in actinomycin‐D clearance assays indicate comparable cytoplasmic stabilities for the two messages. From these results we infer that nuclear prostranscriptional events contribute to the differences in Ldh‐c message levels. © 1993 Wiley‐Liss, Inc.
Effect of sodium and betaine in culture media on development and relative rates of protein synthesis in preimplantation mouse embryos in vitro
Authors: Anbari Kevin , Schultz Richard M.
Published: February 2005

Results of recent experiments indicate that the improved development of mouse embryos in medium containing a low NaCl concentration (85 mM) or the inclusion of the organic osmolyte betaine in a medium containing a high NaCI concentration (125 mM) is correlated with the maintenance of intracellular sodium concentrations that more closely approximate those found in freshly isolated embryos (Biggers et al., 1993, Mol Reprod Dev 34:380–390). We examined the effect of these different culture media on the relative rates of protein synthesis since increased levels of intracellular sodium inhibit protein synthesis; a reduced rate of protein synthesis could therefore account for the differences in development in the different media, since cell division requires protein synthesis. We observe that the ability of these media to support development and to maintain more physiological concentrations of intracellular sodium is correlated with their ability to support increased relative rates of protein synthesis. Reducing the NaCI concentration from 125 mM to 85 mM leads to a greater fraction of the embryos developing from the 2‐cell stage to the 8‐cell stage after 1 day of culture and a substantially improves extent of development to the morula stage after 2 days of culture. This reduction in NaCI concentration also leads to a 2.4‐fold increase in the relative rate of protein synthesis in 4‐cell embryos. Moreover, addition of betaine to medium containing 125 mM NaCI increases the relative rate of protein synthesis. This finding provides an explanation, at least in part, for the increase in development to the blastocyst stage exhibited by mouse embryos cultured in these media. The improved development to the blastocyst is not observed when isolated morulae are cultured in medium containing either a low sodium concentration or in medium containing a high sodium concentration and betaine. These results indicate that the beneficial effect on development of media containing either a low sodium concentration or inclusion of betaine in media containing a high sodium concentration is restricted to stages prior to the morula. © 1993 Wiley‐Liss, Inc.
The specificity of human spermatozoa/zona pellucida interaction under hemizona assay conditions
Authors: Oehninger Sergio , Mahony Mary C. , Swanson James R. , Hodgen Gary D.
Published: February 2005

The objective of this prospective study was to evaluate the specificity of human sperm/zona pellucida interaction under hemizona assay (HZA) conditions in experiments with gametes from the same and different species. Human, cynomolgus monkey and hamster oocytes were used after salt‐storage. Oocytes were bisected into matching hemizonae by micromanipulation and used in the HZA. Semen was obtained from healthy men (donors) and male cynomolgus monkeys and prepared by wash and swim‐up. Sperm binding to matching hemizonae was assessed (tight binding) after 4‐h coincubation in the HZA in homologous and interspecies experiments. Acrosome reaction was evaluated in the sperm droplets using FITC‐PSA and on the hemizonae using the T‐6 monoclonal antibody. On human hemizonae, the number of tightly bound sperm for human and monkey were 93.2 ± 15.8 and 3.9 ± 1.3, respectively (P<0.001). On monkey hemizonae, the number of tightly bound sperm for monkey and human were 126.0 ± 34.8 and 2.8 ± 1.6, (P = 0.02) respectively. On hamster hemizonae, there was negligible binding of human and monkey sperm. There was a significantly higher incidence of acrosome reacted sperm on the zona pellucida in homologous compared to heterologous experiments. These results demonstrate a high species‐specificity of human gamete functions under HZA conditions, providing further support for the use of this bioassay in infertility and contraception testing. © 1993 Wiley‐Liss, Inc.
Combined action of stem cell factor, leukemia inhibitory factor, and cAMP on in vitro proliferation of mouse primordial germ cells
Authors: Dolci Susanna , Pesce Maurizio , De Felici Massimo
Published: February 2005

In the present paper we investigated the effects of stem cell factor/mastocyte growth factor (SCF/MGF), leukemia inhibitory factor/differentiating inhibitory activity (LIF/DIA) (two growth factors known to affect primordial germ cell growth in vitro) and forskolin (FRSK) (an activator of adenylate cyclase in many cell types) alone or in combination on the survival and proliferation of primordial germ cells (PGCs) obtained from 8.5, 10.5, and 11.5 days post coitum (dpc) mouse embryos and cultured without pre‐formed cell feeder layers.

The results showed that both at 1 and 3 days of culture the addition of 100 ng/ml SCF, 20 μM FRSK, or in some instances 20 ng/ml LIF alone caused a significant increase of PGC number as compared with controls. The highest effects were obtained when SCF and/or LIF were used together with FRSK. Moreover, we found that FRSK elevated cAMP levels in purified 11.5 dpc PGCs and that this compound, but not SCF and LIF, stimulated PGC proliferation, as assessed by 5‐bromo‐2′‐deoxyuridin (BrdU) incorporation.

These results suggest a mechanism of combined action of cAMP with SCF and/or LIF in the control of proliferation of mouse PGCs in vitro. © 1993 Wiley‐Liss, Inc.
Glucose, pyruvate, and lactate concentrations in the blastocoel cavity of rat and mouse embryos
Authors: Brison D. R. , Hewitson L. C. , Leese H. J.
Published: February 2005

The concentrations of glucose, pyruvate, and lactate have been measured in the blastocoel fluid of single rat and mouse blastocysts, using the technique of micropuncture combined with an ultramicro‐fluorescence assay. When cultured in the presence of 5.55 mM glucose, 11.5–12.5 mM L‐lactate and 0.25–0.33 mM pyruvate, concentrations in the blastocoel fluid of mouse and rat were 2.30 and 2.75 mM glucose, 14.6 and 19.6 mM L‐lactate, and 0.13 and 0.50 mM pyruvate, respectively. When cultured in the presence of 1.0 mM glucose and 1.0 mM L‐lactate, concentrations in the blastocoel fluid were 0.50 and 0.59 mM glucose and 2.22 and 3.70 mM L‐lactate, respectively. These results suggest that (1) the blastocyst is capable of maintaining considerable concentration gradients of substrates across the trophectoderm, (2) the microenvironment of the blastocoel is adequately supplied with energy substrates for the development of the inner cell mass, and (3) the inner cell mass is capable of developing in both high and low glucose and lactate concentrations. © 1993 Wiley‐Liss, Inc.
Transcriptional activity and nuclear ultrastructure of 8‐cell bovine embryos developed by in vitro maturation and fertilization of oocytes from different growth categories of antral follicles
Authors: Pavlok A. , Kopečný V. , Lucas‐Hahn A. , Niemann H.
Published: February 2005

The normalcy of nuclear differentiation in 8‐cell bovine embryos derived by in vitro procedures from oocytes isolated from antral follicles of three different size categories was studied using autoradiographic localization of RNA synthesis and fine‐structure nuclear morphology. In the few embryos derived from the oocytes from the small category of follicles (1–2 mm) the unlabeled nuclei prevailed. In contrast, the oocytes isolated from the more progressed follicles (medium and large size: 2–8 mm) yielded embryos with only slight, if at all detectable, differences to the picture expected in in vivo developed normal embryos. The diverging features probably concerned a slower onset of gene transcription, its consecutive pattern of localization as well as less pronounced progress of chromatin condensation and nucleolar differentiation. The assessment of these morphological changes and characteristics for individual embryos was obscured by marked differences in the status of the nuclei of particular blastomeres forming an embryo. Here the differences were seen in the fragmentation of nuclei, in the degree of chromatin condensation and in the absence of the nucleolus‐associated chromatin in some of the blastomeres. It is suggested that most of the embryos from medium and large follicles have a comparable differentiation pattern of nuclear function development as embryos developing normally in vivo. © 1993 Wiley‐Liss, Inc.
Analysis of lipid peroxidation mechanisms in human spermatozoa
Authors: Aitken R. J. , Harkiss D. , Buckingham D. W.
Published: February 2005

The mechanisms by which ferrous ion promoters induce malondialdehyde generation by human spermatozoa have been investigated in order to provide a rational basis for the quantification and interpretation of lipid peroxidation assays. Incubation of human spermatozoa with a ferrous ion promoter in the presence of thiobarbituric acid (TBA) led to the generation of the bone fide malondialdehyde‐TBA adduct. The importance of iron in the stimulation of lipid peroxidation was emphasized by the ability of Desferal* and EDTA to suppress malondialdehyde generation. Paradoxically, when the concentration of EDTA relative to iron was equimolar or greater, the suppression of malondialdehyde formation was accompanied by the generation of hydroxyl radicals. These results suggested that the addition of promoter did not effect the first‐chain initiation of lipid peroxidation but favored an alternative mechanism involving the catalytic decomposition of pre‐existing lipid peroxides. This conclusion was reinforced by the inability of reagents that would limit the formation (superoxide dismutase and/or catalase) or availability (mannitol, formate) of hydroxyl radicals, to influence malondialdehyde generation. While hydroxyl radicals were not directly involved in Fe2+‐promoted malondialdehyde generation, the existence of significant correlations between reactive oxygen species production and the outcome of the TBA assay, suggested that Fenton chemistry might be important in the initiation of peroxidative damage. It is proposed that the impeded propagation of peroxidation initiated by Fenton or Haber Weiss reactions would lead to the accumulation of lipid peroxides in the spermatozoa and it is these peroxides that are induced to decompose during the Fe2+‐promoted TBA assay, stimulating a lipoperoxidative chain reaction and malondialdehyde formation. © 1993 Wiley‐Liss, Inc.
Sperm: Egg ratios and putative molecular signals to modulate gamete interactions in polytocous mammals
Authors: Hunter R. H. F.
Published: February 2005

Reasoning from the premises that 1) the sperm:egg ratio at the time of activation of the secondary oocyte in mammals is close to unity under conditions of spontaneous mating, 2) a majority of eggs within the cumulus oophorus of a polytocous species is fertilized in a reasonably short interval of time, and 3) spermatozoa would find it difficult to reverse their approach to the zona pellucida, it is proposed that molecular gradients exist to divert spermatozoa penetrating the cumulus mass away from eggs already activated and towards eggs as yet unfertilised. Possible sources of such molecular cues are considered, as is the event that triggers their release. © 1993 Wiley‐Liss, Inc.
The IGFs and their binding proteins in murine development
Authors: Murphy Liam J. , Barron Douglas J.
Published: February 2005

Recent observations suggest that the diverse actions of the insulin‐like growth factors (IGFs) are the result of interactions of the various components that make up the IGF system. The components of this system include IGF‐I and ‐II and their variants, the type 1 and 2 IGF receptors and the insulin‐like growth factor binding proteins (IGFBPs). Various components of the IGF system are expressed in the developing mouse embryo and the adjacent tissues of the reproductive tract in which the embryo develops. Thus there is the potential for paracrine interactions between the maternal and fetal tissues. Transcripts for the IGF receptors, IGF‐I and IGF‐II, have been demonstrated in the periimplantation mouse embryo. While there are now data from gene ablation experiments indicating that IGF‐II is important in embryogenesis, the role of other components of the IGF system such as the IGFBPs remains unclear. The data accumulated so far are largely empirical, and there is as yet little compelling evidence that maternal IGFs derived from oviduct or uterine fluid and maternal tissues are necessary for normal fetal development. We have started to develop transgenic mice lines overexpressing IGFBPs to attempt to address the role of these binding proteins in fetal development. © 1993 Wiley‐Liss, Inc.
Transcriptional regulation of the major promoters of the human IGF‐II gene
Authors: Holthuizen P. , Van Dijk M. A. , Rodenburg R. J. T. , Koonen‐Reemst A. M. C. B. , Sussenbach J. S.
Published: February 2005

The human insulin‐like growth factor‐II (IGF‐II) gene constitutes a complex transcriptional unit that is controlled by four different promoters preceding multiple 5′‐untranslated exons. Expression of the IGF‐II gene is development and tissue specific and yields a family of mRNAs that all encode preproIGF‐II. We have analyzed the sequences and factors involved in the transcriptional activation of promoters P1 and P3. Detailed analysis revealed several characteristic elements and protein binding sequences. P1, which is expressed only in adult liver tissue, contains a region that can be specifically activated by the CCAAT/enhancer binding protein (C/EBP). P3 consists of a proximal promoter region that supports general transcription, whereas cell‐specific elements are located upstream. © 1993 Wiley‐Liss, Inc.
The IGF‐II receptor system: A G protein‐linked mechanism
Authors: Nishimoto Ikuo
Published: February 2005

Based on the finding that stimulation of the IGF‐II, receptor (IGF‐IIR) is capable of activating Gi2 and calcium channels in BALB/c 3T3 fibroblasts, it was found that purified IGF‐IIR can couple directly to purified Gi2 in phospholipid vesicles. IGF‐IIR–Gi2 coupling can be characterized as follows. IGF‐IIR directly couples to Gi2 in response to IGF‐II in a stoichiometrical manner, suggesting that IGF‐IIR works as a transmembrane signaling molecule and that the seven‐transmembrane structure is not essential for receptor‐G protein coupling. The mode of IGF‐IIR–Gi2 interaction is similar to that of conventional receptor–G protein coupling, suggesting that a common G protein recognition mechanism is shared by IGF‐IIR and conventional G‐coupled receptors. The action of IGF‐IIR is specific on Gi2 among various G proteins. Finally, the activity of IGF‐IIR on Gi2 is similarly potent across the species of the proteins. These characteristics led to the discovery of a 14‐amino‐acid region in IGF‐IIR that can directly interact with and activate Gi2, and is located at residues 2410–2423 of the human receptor. Subsequent work has indicated that this region is responsible for Gi‐coupling function of intact IGF‐IIR. The most important extensions of this discovery are the following: (1) The structure–function relationship for the Gi‐activating function of this 14‐amino‐acid sequence, (2) the prediction of G protein‐coupled functions of receptors based on the results obtained from 1), and (3) clarification of the detailed mechanism whereby ligand–receptor complex recognizes G proteins. This paper reviews what we have learned from IGF‐IIR in terms of receptor–G protein interfaces and discusses future prospects. © 1993 Wiley‐Liss, Inc.
IGF‐I and the IGF‐I receptor in development of nonmammalian vertebrates
Authors: De Pablo Flora , Pérez‐Villamil Beatriz , Serna José , González‐Guerrero Pilar R. , López‐Carranza Ana , De La Rosa Enrique J. , Alemany Jorge , Caldés Trinidad
Published: February 2005

Extracellular signals are likely to be involved in the control of growth and differentiation during embyrogenesis of vertebrates. These signals include, among others, several members of the insulin family: insulin‐like growth factor (IGF)‐I, IGF‐II, and insulin. In the chick embryo, maternal IGF‐I is stored in the yolk. In addition, the embryonic IGF‐I gene is expressed very early and in late development in multiple tissues. We have used reverse‐transcribed (RT) RNA and amplification by the polymerase chain reaction (PCR) to detect IGF‐I gene expression. IGF‐I was preferentially expressed in cephalic regions during late neurulation and early organogenesis. During late organogenesis, in some tissues, such as the eye lens, IGF‐I gene expression is compartmentalized to a subset of cells, the epithelial cells. In these lens cells, IGF‐I stimulates transcription of the δ‐crystallin gene. Competence to respond to IGF‐I exists in multiple cell types, since, based on binding studies, receptors for IGF‐I are widespread in the gastrulating and neurulating embryo. Target tissues in which an autocrine/paracrine role for IGF‐I appears more likely are the developing eye lens and retina, which are avascular organs rich in IGF‐I receptors. In late development, IGF‐I may have an additional endocrine role, with an impact on the general growth of the chick embryo. In embryos developed ex ovo, that show growth retardation after day 10 of embyrogenesis, IGF‐I serum levels are very low. By day 8, expression of IGF‐I mRNA in these embryos is markedly reduced in multiple tissues. Future studies in which IGF‐I and its receptor are overexpressed or abolished should clarify the function of this growth factor in development. © 1993 Wiley‐Liss, Inc.
Cell allocation in twin half mouse embryos bisected at the 8‐cell stage: Implications for preimplantation diagnosis
Authors: Hardy Kate , Handyside Alan H.
Published: February 2005

Previous work in the mouse and human has shown that removal of cells from 2‐ and 4‐cell embryos disproportionately reduces the inner cell mass (ICM), from which the fetus is derived, suggesting that preimplantation biopsy at these stages would be inadvisable. Here we have examined allocation in twin half mouse embryos produced by bisection at the 8‐cell stage. Half embryos and intact zona‐free control embryos were differentially labeled at the time of cavitation using polynucleotide‐specific fluorochromes, and the numbers of cells in the ICM and trophectoderm (TE) counted. Adding the numbers of cells in the twin halves together, the numbers of ICM cells are not significantly different from those in intact controls. In contrast, numbers of TE cells were significantly higher (P < 0.01), producing a small increase in the total cell number of half embryos. There is no concomitant decrease in ICM cell number. We propose that as the surface area of a half embryo is greater than half an intact control, TE cells in half embryos divide faster in order to reach an optimal packing density similar to that in intact control embryos. Furthermore, we suggest that preimplantation biopsy only be performed at the 8‐cell stage or later. © 1993 Wiley‐Liss, Inc.
Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution, and development in nuclear transplant bovine embryos
Authors: Barnes F. L. , Collas P. , Powell R. , King W. A. , Westhusin M. , Shepherd D.
Published: February 2005

Nuclear transplantations into metaphase II (MII) and S phase oocyte cytoplasm were performed to investigate the influence of recipient cell cycle stage on nuclear function and development of bovine nuclear transplant (NT) embryos. Rate of inactivation of histone H1 kinase and duration of DNA synthesis in activated oocytes were determined. The proportion of S phase blastomeres in in vivo produced day 5.5 bovine embryos was measured. DNA synthesis was also assessed in NT embryos after transfer into MII and S phase cytoplasm. MII NT embryos were produced by fusing a blastomere into a MII oocyte; the fusion pulse served to activate the oocyte. S NT embryos were produced by fusing a blastomere into an early S phase oocyte electrically activated 4 h prior to fusion. Nuclear envelope structure, chromosome constitution, and extent of development were examined in MII and S NT embryos. Histone H1 kinase activity dropped to baseline within 2 h of electrical activation. A second electrical pulse did not alter H1 kinase activity when delivered 4 h after the first pulse. The frequency of S phase blastomeres in day 5.5 bovine embryos ranged from. 79% to 100%, depending on the duration of culture in 3H‐thymidine. Nuclear transplantation into MII cytoplasm resulted in a transient drop in DNA synthesis over 3.5 h. DNA synthesis resumed at 4.5 h post activation (hpa), concomittantly with initiation of DNA replication in activated oocytes. In contrast, DNA synthesis was not interrupted after transfer into S phase cytoplasm. DNA synthesis persisted until 13.5 hpa, as in activated oocytes. Partial or complete nuclear envelope breakdown (NEBD) occurred after transfer into MII cytoplasm, whereas the nuclear envelope remained intact in 50% of the embryos or underwent partial breakdown in S phase cytoplasm. A greater proportion of S NT embryos was diploid (50% vs. 23% MII NT embryos, P < 0.001), and a higher frequency of S NT embryos developed to the morula or blastocyst stage (22% vs. 5%, P < 0.001). The data indicate that DNA synthesis is regulated differently if the recipient oocyte is in MII or in S phase at the time of fusion. Extended DNA synthesis after transfer into MII cytoplasm suggests a re‐replication of the donor chromatin. Re‐replication, presumably, does not occur after transfer into S phase cytoplasm. Re‐replication is likely to be a consequence of permeabilization of the nuclear envelope upon NEBD in MII cytoplasm. Improved regulation of DNA synthesis after transfer into S phase cytoplasm and reduced incidence of chromosome damage in the first cell cycle may have been responsible for increased frequency of development of S NT embryos to the morula/blastocyst stage. © 1993 Wiley‐Liss, Inc.