bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin regulates protein metabolism in mouse blastocysts
Authors: Dunglison Greta F. , Kaye Peter L.
Published: February 2005

Mouse blastocysts, in vitro, endocytosed 100 μg/ml 125I‐labelled bovine serum albumin (BSA) at a rate equivalent to 192 ± 27 μl/hr/mg embryonic protein over the first 20 min. Insulin stimulated this initial uptake by 30% (P < 0.05). After this time, accumulation of 125I‐labelled BSA began to plateau as the endocytosed 125I‐labelled BSA was catabolized and 125I was released from the cells. Insulin caused an ≈︁72% (P < 0.05) increase in the amount of uncatabolized 125I‐labelled BSA remaining in insulin‐treated blastocysts after 2 hr as compared to control blastocysts. Insulin partially inhibited catabolism of endocytosed 125I‐labelled BSA during the first 2 hr following transfer to nonradioactive medium. After this time, degradation ceased in both control and insulin‐treated blastocysts, leaving a small, uncatabolized protein pool remaining in the embryos; however, as a result of insulin's inhibitory effects on the initial catabolic rate, the uncatabolized protein pool was 30% (P < 0.05) larger in insulin‐treated blastocysts after the 4 hr chase. Insulin inhibited endogenous protein degradation in blastocysts by 37% (P < 0.05). Combined with previous studies showing a 90% increase in endogenous protein synthesis in blastocysts following short‐term stimulation with insulin (Harvey and Kaye, 1988), these results suggest that insulin acts to increase the endogenous protein‐reserves in the embryo. Dose‐response studies indicated an EC50 of 0.5 pM for insulin's stimulation of 125I‐labelled BSA accumulation, consistent with action via its own receptor. Insulin‐like growth factor‐1 (IGF‐1) also stimulated protein accumulation at concentrations similar to those observed with insulin, suggesting that IGF‐1 may act via its own receptor rather than the insulin receptor to exert its effects on endocytosis. © 1993 Wiley‐Liss, Inc.
Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte‐cumulus cell complexes in vitro
Authors: Singh Balwant , Barbe Gerald J. , Armstrong David T.
Published: February 2005

The present study was undertaken to examine effects of various combinations of epidermal growth factor (EGF), transforming growth factor‐b̃1 (TGF‐b̃1), follicle‐stimulating hormone (FSH), luteinizing hormone (LH), androstenedione (A4), and estradiol‐17b̃ (E2) on meiotic maturation and cumulus expansion in the pig using an in vitro model system. Oocyte‐cumulus cell complexes (OCC) were cultured in the media containing the abovementioned agents for 24 hr and were observed for germinal vesicle breakdown (GVBD), indicative of initiation of meiotic maturation, and for expansion of their cumulus cells. Treatment with EGF significantly increased (P < 0.05) incidence of GVBD, with maximal stimulation occurring at 1 ng/ml (55% vs. 12% in the control). Concentrations of EGF as low as 100 pg/ml significantly stimulated GVBD over control (37% vs. 12%). Addition of EGF (1 ng/ml) and FSH (1.5 μg/ml) together and LH (2 μg/ml) and FSH (1.5 μg/ml) together resulted in significantly higher (P < 0.01) GVBD levels than were observed in response to EGF, FSH, or LH alone. Addition of E2 (1 μg/ml) had no effect by itself but significantly decreased the incidence of GVBD in the presence of FSH and of LH + FSH. Addition of A4 (1 μg/ml) significantly reduced the percentage of oocytes undergoing GVBD when added alone or with FSH. Although both EGF and LH stimulated cumulus expansion, FSH was more effective in stimulating cumulus expansion than EGF or LH. TGF‐b̃1 had no effect on GVBD or cumulus expansion. These studies indicate that these hormones may have differing roles in oocyte maturation and that their interactions may be part of an intricate system regulating the maturation of oocytes during follicular development in vivo. © 1993 Wiley‐Liss, Inc.
Embryonic stem cells derived from morulae, inner cell mass, and blastocysts of mink: Comparisons of their pluripotencies
Authors: Sukoyan Marina A. , Vatolin Sergei Y. , Golubitsa Alevtina N. , Zhelezova Antonina I. , Semenova Lora A. , Serov Oleg L.
Published: February 2005

A characterization of cell lines that we derived from morulae (three lines), blastocysts (two lines), and the inner cell mass (ICM) is given. The karyotype of all the lines was normal; the genotype of four lines was XX, and four lines were genotypically XY. The pluripotencies and commitment status of the derived lines were estimated. First, there were not less than two‐thirds of cells in the populations of the lines derived from morulae and the lCM with both Xs active; 70–100% of cells of the blastocyst‐derived lines had one of the Xs in an inactive state. The activity of glucose‐6‐phosphate dehydrogenase (G6PD) in the lines (genotype XX) derived from morulae and ICM was found to be twofold higher than in lines with genotype XY, and G6PD activity was the same in the blastocyst‐derived XX lines and XY lines. Second, when injected intraperitoneally into athymic mice, morulae‐ and ICM‐derived cells gave rise to simple and complex embryoid bodies (EB) resembling to typical “cystic” mouse EBs. Third, when injected subcutaneously to athymic mice, the ICM‐ or morula‐derived cells gave rise to typical teratomas containing derivatives of the three germ layers and components of organogenesis. Comparisons of cell lines of different derivations demonstrated that the pluripotencies of the ES cells derived from morulae or the ICM are higher than those of blastocyst derivation. © 1993 Wiley‐Liss, Inc.
Evidence for the production of a growth‐inhibitory factor by human granulosa‐luteal cells
Authors: Dain Liliana B. , Bley Miguel A. , Barañao José L. , Tesone Marta
Published: February 2005

The factors involved in the inhibition of ovarian follicular cellular growth after the luteinizing hormone (LH) surge are poorly established. The aim of this study was to investigate the production of an inhibitory growth factor by human ovarian cells. Luteinized granulosa cells were obtained from an assisted fertilization program and were cultured in the presence or absence of follicle‐stimulating hormone (FSH) and estradiol. Data obtained by cell counting showed that the number of human luteinized granulosa cells cultured in the presence of fetal bovine serum (10%) increased 1.8‐fold within a 2‐day period. In serum‐free medium, human luteinized granulosa cells were able to incorporate 3H‐thymidine, measured during consecutive 48 h periods. During all the periods tested (up to 7 days), low basal levels of thymidine incorporation were measured and were further reduced in the presence of FSH (200 ng/ml) and estradiol (500 ng/ml). To elucidate the possible production of an inhibitory growth factor, 3H‐thymidine incorporation by rat granulosa cell cultures was measured in the presence of conditioned media (CM; from human granulosa cell cultures). In this system, FSH and estradiol elicited a tenfold increase in thymidine incorporation. The addition of CM (10% v/v collected on day 2) to FSH‐ and estradiol‐treated granulosa cell cultures produced an inhibition (61%) of thymidine incorporation. The active factor in CM withstood freeze‐thawing, was stable for several weeks at – 20°C, became unstable at 4°C, and was heat labile and sensitive to proteolysis. Ultrafiltration using membranes with different molecular weight cutoffs suggested that the factor had a molecular weight >30,000 dalton. We suggest that an inhibitory growth factor produced by human luteinized granulosa cells could be involved in the differentiation of growing follicles to corpus luteum. © 1993 Wiley‐Liss, Inc.
Nuclear transfer and electrofusion in bovine in vitro‐matured/in vitro‐fertilized embryos: Effect of media and electrical fusion parameters
Authors: Van Stekelenburg‐Hamers Annelies E. P. , Van Inzen Wouter G. , Van Achterberg Tanja A. E. , Kruip Theo A. M. , De Laat Siegfried W. , Weima Sjerp M.
Published: February 2005

In this study, micromanipulation and electrofusion conditions for the cloning of in vitro‐produced bovine embryos (here after termed IVM/IVF embryos) derived from in vitro‐matured (IVM) and in vitro‐fertilized (IVF) oocytes were established. The effect of DC field strength on the fusion rate was tested in a model system using pronuclear stage embryos in which a cytoplasmic vesicle was removed and reinserted. Efficient fusion (80%) was obtained by applying a pulse of 1.75 kV/cm for 40 μsec. In vitro development of manipulated pronuclear stage embryos was as efficient as that of unmanipulated control embryos. Different fusion media were compared in the cloning procedure, using IVM oocytes as recipients and blastomeres from day 6 IVM/IVF donor embryos. Zimmermann cell fusion medium reduced the lysis of nuclear transfer embryos compared to F300 (5% vs. 25%). The effects of drugs disrupting the microfilaments and microtubuli were determined. Neither the addition of cytochalasin B (CCB) for 1 hr in the postfusion medium nor incubation of donor blastomeres with nocodazole had a significant effect on the fusion or cleavage rate of the nuclear transfer embryos. Additional experiments demonstrated that there was no difference in developmental potential between nuclear transfer embryos allowed to develop in vitro or in vivo and that the embryos gave a 15% pregnancy rate in recipient cattle. © 1993 Wiley‐Liss, Inc.
Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction
Authors: Gutiérrez A. , Garde J. , García‐Artiga C. , Vázquez I.
Published: February 2005

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS‐2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell‐conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO2 in air. Viability and the occurrence of true acrosome reactions were assessed using a triple‐stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS‐2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS‐2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS‐2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley‐Liss, Inc.
Relationships between the completion of first cleavage and the chromosomal complement, sex, and developmental rates of bovine embryos generated in vitro
Authors: Yadav B. R. , King W. A. , Betteridge K. J.
Published: February 2005

One thousand eighty‐four two‐cell bovine embryos produced from 1,574 oocytes matured and fertilized in vitro were cultured as groups separated according to the time when they completed their first cleavage (24,30,40,48, or 62 hr postinsemination; hpi). At 5 days after insemination, the proportions of each group that had progressed to the eight‐cell stage or beyond were determined and the 350 that had done so were fixed and examined cytogenetically for cell number, chromosomal abnormalities, and sex. Embryos in the “early” cleaving (24 and 30 hpi) and “late” cleaving (40–62 hpi) groups were compared. Early cleaving embryos were more likely to have developed to the eight‐cell stage or beyond (52.2% vs. 20%), contained more cells (22 vs. 17), and were more likely to be male (3.6:1 vs. 0.93:1). It is suggested that these phenotypic differences between the sexes begin before the embryonic genome is generally thought to become activated and are due either to differential processing of X‐ and Y‐bearing sperm within the zygote or to very early differential expression of genes derived from X‐ and Y‐bearing sperm. © 1993 Wiley‐Liss, Inc.
Video microscopic analysis of ionophore induced acrosome reactions of lobster (Homarus americanus) sperm
Authors: Tsai K. L. , Talbot P.
Published: February 2005

Sperm from the American lobster (Homarus americanus) are normally nonmotile. However, during fertilization, the sperm undergo a calcium‐dependent acrosome reaction that propels them forward about 18 μMm. The reaction occurs in two phases, eversion and ejection, which take place too quickly to permit analysis by direct observation. The purposes of this study were to examine the structural changes occurring in sperm during the normal acrosome reaction and to determine the rate of the reaction using video microscopy. The reaction was induced in vitro by ionophore A23187 and recorded using a video system attached to a Nikon Nomarski interference microscope. Videotapes were played back frame by frame (30 frames/sec), and images of reactions from 10 sperm were analyzed. The acrosome reaction, including the eversion of the acrosomal vesicle and ejection of the subacrosomal material and nucleus, can be divided into 4 steps: (1) expansion of the apical cap followed by expansion of the remainder of the acrosomal cylinder; expansion of the cylinder begins at its apical end and proceeds toward its base, (2) eversion of the apical half of the acrosomal vesicle and initial contraction of the apical cap, (3) eversion of the basal half of the acrosomal vesicle, continued contraction of the apical cap, and ejection of the subacrosomal material and nucleus, and (4) final contraction of the apical cap and ejection of the acrosomal filament. During steps 2, 3, and 4, the mean forward movement of sperm is 12.7, 3.9, and 1.1 μMm, respectively. Although the time required to complete the reaction ranged from 0.66 to 5.16 s, most sperm reacted in less than 3. s, and these sperm were considered to have typical rates. For sperm that reacted in less than 3 s, both step 1 and step 4 take about 0.2 s and show little variation among sperm. the time required to complete steps 2 and 3 averaged 0.63 and 0.37 s, respectively. Forward movement of the sperm during the acrosome reaction is caused by eversion of the inner and outer acrosomal material and contraction of the apical cap. The protein(s) responsible for this contraction is not yet known. © 1993 Wiley‐Liss, Inc.
Chromatin condensation and acrosome development during spermiogenesis of Ensis ensis (Mollusca, Bivalvia)
Authors: Casas M. T. , Subirana J. A.
Published: February 2005

We describe chromatin condensation and acrosome development during spermiogenesis of Ensis ensis. The overall shape of the mature spermatozoon corresponds to the primitive type. The nucleus is oval and on its superior pole there is an elongated acrosome; the middle piece contains four mitochondria around the centriolar complex. The condensation of the nuclei seems to occur in three steps: first the diameter of chromatin fibers increases slightly from 17 to 20 nm; second, in midspermatids fiber pairs coalesce; and third, the coalescence continues by addition of other fibers until the nuclei become highly compacted. Chromatin changes are related with nuclear protein composition. Small proacrosomal vesicles show two regions of different electron density. At a later stage they fuse to give a single, spherical vesicle in round spermatids, which migrates to the upper pole and transforms into a tapered acrosome (18 μm long) with a central channel filled with finely fibrous material. © 1994 Wiley‐Liss, Inc.
Functions of milk protein gene 5′ flanking regions on human growth hormone gene
Authors: Ninomiya Takashi , Hirabayashi Masumi , Sagara Junko , Yuki Atsushi
Published: February 2005

Fragments containing 5′ flanking regions of four bovine milk protein genes—alpha lactalbumin (bαLA), alpha S1 casein (bαS1CN), beta casein (bβCN), kappa casein (bkCN)—and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5′ regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two‐thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying bαLA/, bβCN/, and mWAP/hGH, and 33% of the laclated bαS1CN/hGH rats secreted detectable amounts of hGH (> 0.05 μg/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of bαLA/, bβCN/, bαS1CN/, and mWAP/hGH rats were 1.13–4,360 μg/ml, 0.11–10,900 μg/ml, 86.8–6,480 μg/ml, and 6.87–151 μg/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 μg/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen. None of the bαCN/hGH rats secreted detectable amount of hGH into their milk, whereas 8 of the 11 lines secreted hGH into their sera. For the production of hGH in transgenic rat milk, the 5′ region of bαS1CN was shown most suitable, because the bαS1CN/hGH rat secreted > 6,000 μg/ml of hGH into the milk and could be reproduced. © 1994 Wiley‐Liss, Inc.
Cloning and tissue expression of bovine follistatin cDNA
Authors: Houde A. , Lussier J. G. , Ethier J.‐F. , Gagnon C. , Silversides D. W.
Published: February 2005

Bovine follistatin cDNA sequences were derived using a cloning strategy based entirely on reverse transcription and polymerase chain reaction amplification of RNA derived from bovine ovarian and testicular tissues. Complete bovine follistatin cDNA coding sequences are presented including 1,029 bases of open reading frame, the 5′ translational start codon, and the 3′ translational stop codon. Homologies of bovine follistatin cDNA with pig, human, rat, and partial sheep sequences are 94.3%, 92.4%, 89.9%, and 98.4% at the nucleic acid level and 98.3%, 97.1%, 95.6%, and 100% at the deduced amino acid level, respectively. Northern blot analysis on a survey of bovine reproductive tissues showed strongest expression in ovaries collected from superovulated cows and major RNA species at 2.8 Kb and 1.75 Kb. © 1994 Wiley‐Liss, Inc.
Identification of anti‐agglutinin for spermatozoa in epididymal boar plasma
Authors: Harayama H. , Miyano T. , Miyake M. , Kusunoki H. , Kato S.
Published: February 2005

The present report identifies epididymal boar anti‐agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs–Ringer bicarbonate at 37°C (5% CO2 in air). In the samples washed three or five times and then incubated for 3–5 h, higher rates (72–79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2–5 cells (head‐to‐head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head‐to‐head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti‐agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti‐agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro. © 1994 Wiley‐Liss, Inc.
Characterization of mouse‐hamster‐human cross‐reacting antioocyte monoclonal antibodies produced by intrasplenic immunization of mice with 12 zona‐free mouse oocytes
Authors: Jin Meishan , Johansson Lars , Sundstrōm Per , Nilsson B. Ove
Published: February 2005

Several intrasplenic immunizations with batches of ∼15 or ∼30 zona‐free, unfertilized mouse oocytes resulted in 200–300 hybrids, respectively, among which about 20 positive clones were selected from each fusion between splenic plasma cells and SP2/0 myeloma cells. When nonimmunized splenic plasma cells were used, only one antibody, showing weak immunoreaction, was obtained from ∼370 hybrids collected from 2 fusions. From one immunization with a total of 12 zona‐free, unfertilized mouse oocytes, 15 positive clones were selected for further study. Eleven of these 15 antibodies reacted with antigens only in unfertilized oocytes but not in fertilized, pronuclear stage oocytes. Three antibodies, which recognized antigens in paraffin‐embedded oocyte sections, did not label growing ovarian oocytes, indicating that the antibodies were specific to ovulated, unfertilized oocytes. These antibodies did not detect any antigen epitopes in the panel of tissues examined. The molecular weight of one antigen, corresponding to a IgM antibody that is present both in ooplasma and zona pellucida, was ∼116 kDa. Cross‐reactivity to blots of unfertilized zona‐free hamster oocytes was demonstrated by 6 antibodies and to unfertilized human oocytes by 7 antibodies. Three antibodies cross‐reacted with both hamster and human oocytes. The study indicates that the intrasplenic immunization is an appropriate means of raising antibodies against unfertilized, zona‐free mouse oocytes and that the method applied offers an easy way to select antibodies against human oocytes for functional studies. © 1994 Wiley‐Liss, Inc.
Calmodulin during human sperm incorporation into hamster oocyte: An immunogold electron microscope study
Authors: Courtot Anne‐Marie J. , Feinberg Jacqueline M. , Schoevaert Damien A. , Rainteau Dominique P. , Weinman Serge J.
Published: February 2005

In the present study, immunogold labeling of ultrathin sections of human sperm, before and after incorporation into hamster oocyte, was used to obtain insight into the ultrastructural localization and possible function of calmodulin during fertilization. In heads of ejaculated, capacitated, and acrosome‐reacted fixed human sperm, calmodulin was mainly found in two compartments, the subacrosomal layer and the postacrosome. After sperm‐egg fusion, the subacrosomal calmodulin was unaltered and surrounded by the fertilization cone in which actin was abundant. There was no co‐localization of calmodulin and actin. In contrast, postacrosomal calmodulin disappeared as soon as the sperm head was incorporated into egg cytoplasm. These unique localizations and redistributions are in agreement with the concept of a calmodulin targeting from acrosome toward postacrosome through the subacrosomal layer during spermatogenesis (Weinman et al., 1986b: J Histochem Cytochem 34:118). Moreover, they strongly suggest a role for calmodulin both in sperm‐egg fusion and in the initial pulse of Ca2+ occurring during fertilization. © 1994 Wiley‐Liss, Inc.
Stimulating effect of pyroglutamylglutamylprolineamide, a prostatic, TRH‐related tripeptide, on mouse sperm capacitation and fertilizing ability in vitro
Authors: Green Catherine M. , Cockle Sheena M. , Watson Paul F. , Fraser Lynn R.
Published: February 2005

Pyroglutamylglutamylprolineamide, a prostatic tripeptide with structural similarities to thyrotrophin‐releasing hormone (TRH), has been found in the seminal plasma of several mammalian species, suggestive of a biological function relating to spermatozoa. Using chlortetracycline (CTC) fluorescence analysis and in vitro fertilization, we have obtained evidence that the tripeptide stimulates mouse sperm capacitation and fertilizing ability in vitro. The tripeptide at concentrations from 5–500 nM was added to sperm suspensions and cells were assessed with CTC after 40 min, insufficient time for complete capacitation by a majority of spermatozoa under standard conditions of incubation. Concentrations of 25 nM and higher significantly promoted capacitation, as evidenced by a decrease in the proportion of acrosome‐intact F pattern spermatozoa, characteristic of uncapacitated cells, and an increase in the proportion of acrosome‐intact B pattern spermatozoa, characteristic of capacitated cells. However, there was no significant stimulation of acrosomal exocytosis. These results suggested that peptide‐treated cells would be more fertile than their untreated counterparts. This was confirmed using in vitro fertilization, where the presence of 100 nM peptide during sperm preincubation and gamete coincubation significantly stimulated fertilizing ability (peptide, 56.5% of oocytes fertilized; controls, 26.5%). Comparison of the prostatic tripeptide and TRH effects on capacitation revealed that TRH at a concentration of 250 nM was as effective as the prostatic tripeptide in promoting the F & B transition but was less effective or ineffective at lower concentrations. In vitro fertilization assessment of the two peptides, at 100 nM, revealed that only the prostatic tripeptide significantly stimulated fertility. Again, this was consistent with the CTC analyses. Because the prostatic tripeptide can stimulate sperm function in vitro, it is possible that it plays a similar role in vivo and promotes fertilizing ability of ejaculated spermatozoa. We therefore propose that this tripeptide be referred to as fertilization promoting peptide (FPP). © 1994 Wiley‐Liss, Inc.
Evidences for the presence of chymotrypsin‐like activity in human spermatozoa with a role in the acrosome reaction
Authors: Morales Patricio , Socias Teresa , Cortez Jacqueline , Llanos Miguel N.
Published: February 2005

The effect of chymotrypsin inhibitors and substrates on the human sperm acrosome reaction stimulated by the human zonae pellucidae or follicular fluid were evaluated. Motile spermatozoa, selected by a Percoll gradient, were incubated at 1 × 107 cells/ml, 37°C, and 5% CO2, After 4.5 hr, the chymotrypsin inhibitor TPCK (N‐Tosyl‐L‐Phenylalanine‐Chloromethyl Ketone) or the substrate ATEE (N‐Acetyl‐L‐Tyrosine Ethyl Ester) were added for 30 min. Then, four oocytes were added and the percentage of acrosome‐reacted spermatozoa on the zona was determined. TPCK and ATEE inhibited the zona pellucida‐induced acrosome reaction. The chymotrypsin inhibitors TPCK and chymostatin and the chymotrypsin substrates ATEE, BTEE (N‐Benzoyl‐L‐Tyrosine Ethyl Ester), Succinyl‐Ala‐Ala‐Phe‐7‐Amido‐4‐Methyl‐Coumarin (Suc‐Ala‐Ala‐Phe‐AMC), and Succinyl‐Leu‐Leu‐Val‐Tyr‐7‐Amido‐4‐Methyl‐Coumarin (Suc‐Leu‐Leu‐Val‐Tyr‐AMC) inhibited the human follicular fluid‐induced acrosome reaction. Sperm extracts exhibited hydrolytic activity toward Suc‐Ala‐Ala‐Phe‐AMC and Suc‐Leu‐Leu‐Val‐Tyr‐AMC. This enzyme activity was abolished by TPCK and chymostatin, was independent of Ca2+, and was not modified by 1,10 phenanthroline. In addition, the activity was present in the supernatant after the acrosome reaction was induced with calcium ionophore and in epididymal spermatozoa recovered from the cauda region. Electron microscopic observations indicated that the inhibitors prevented the membrane events of the acrosome reaction. These data suggest an association between human spermatozoa and chymotrypsin‐like activity with a possible role in the acrosome reaction. © 1994 Wiley‐Liss, Inc.
Analysis of mRNA for class I HLA on human gametogenic cells
Authors: Janitz Michal , Fiszer Dorota , Michalczak‐Janitz Karolina , Lukaszyk Andrzej , Fernandez Nelson , Skorupski Witold , Kurpisz Maciej
Published: February 2005

We have studied mRNA expression for Class I HLA (human leukocyte antigen) on male germ cells by amplification of gene fragments in PCR techique and by Northern hybridization. RNA was extracted from fractionated gametogenic cells (isolated from testis) and reversely transcribed. Then, cDNA was amplified for specific HLA sequence (1151 bp) representing whole‐length coding sequence (HLA, ‐A, ‐B, ‐C). The specificity of this product was confirmed in “nested” PCR of 400 bp gene fragment coding for alpha 2 domain, alpha 3 domain, and the transmembrane portion of Class I HLA. The results indicate minimal expression of classical Class I HLA on gametogenic cells. Northern hybridization with 669 bp cDNA fragment (spanning for alpha 3 domain, transmembrane, cytoplasmic, and 3′ untraslated region) resulted in a low intensity signal from gametogenic cell fractions and confirmed our findings obtained by PCR. The minimal expression of classical HLA antigens may create a neutral cover for the male reproductive system, thereby preventing an immunological response during germ cell differentiation. © 1994 Wiley‐Liss, Inc.
Expression of the imprinted gene H19 in the human fetus
Authors: Lustig O. , Ariel I. , Ilan J. , Lev‐Lehman E. , De‐Groot N. , Hochberg A.
Published: February 2005

The H19 gene is a parenterally imprinted maternally expressed gene which has a pivotal role in embryogenesis and fetal development. It is tightly linked to the IGF‐II gene on chromosome 11p15.5 which is reciprocally imprinted. We studied the expression of the human H19 by in situ hybridization in an embryo 35 days post coitus (dpc) and in a fetus from the second trimester of pregnancy. The expression pattern of H19 in the human fetal tissues was similar to its expression in the mouse, and paralleled, with some exceptions, the expression of IGF‐II in human fetuses. Abundant expression was found in organs comprising the fetoplacental unit: the placenta, the fetal adrenal, and liver. The expression in the fetal adrenal cortex was most prominent in the definitive cortex and somewhat weaker in the fetal zone. Considerable expression of H19 was found in the fetal liver as early as 35 dpc and in the second trimester. Hematopoietic cells in fetal liver did not express the gene. Moderate expression of H19 was detected in the epithelium of the small intestines, in endometrial stroma and Fallopian tube. In the kidney conspicuous labeling of the metanephric blastema was noted, which was markedly reduced with differentiation to tubules. This pattern of expression is identical to that of IGF‐II in the fetal kidney and is relevant to the evolution of Wilms' tumor. No expression of H19 was found in the neural tube of the first trimester embryo or in the developing fetal brain in the second trimester, nor were transcripts detected in the choroid plexus. The localization of H19 expression within human fetal tissues may help to elucidate its role in human embryogenesis and fetal development. © 1994 Wiley‐Liss, Inc.
Maturation of hamster epididymal sperm motility and influence of the thiol status of hamster and rat spermatozoa on their motility patterns
Authors: Yeung C. H. , Oberländer G. , Cooper T. G.
Published: February 2005

A method for objective quantification of hamster sperm movement parameters as an indicator of maturation along the epididymis was established using a computerised system. Analysis of spermatozoa released into medium from five epididymal regions showed that the most drastic increases in percentage motility and curvilinear velocity (VCL) occurred from the distal corpus to the beginning of the proximal cauda and in straight‐line velocity (VSL) from the beginning to a more distal site within the proximal cauda region. Both high osmolarity (400 mOsm/kg) and the thiol‐oxidising agent diamide (10 μM) increased flagellar straightness of distal corpus spermatozoa, but VSL was increased only with the latter. The thiol‐reducing agent dithiothreitol (DTT, 1mM) stimulated and maintained percentage motility and velocities of spermatozoa from the caput, stimulated only percentage motility of distal corpus sperm, but decreased velocities of those from the proximal cauda in prolonged incubation. In rats, diamide increased path straightness but not velocities of caput spermatozoa and yet caused immotility within 15 min, whereas DTT prolonged the maintenance of in vitro motility. The slight increases in kinematic parameters in the presence of DTT were enhanced by a 2‐min preincubation with diamide. The finding that the effects of DTT and diamide were not compensatory suggests that the influence of the SH/S‐S status on sperm movement is multifaceted, with decreasing sensitivity to stimulation upon sperm maturation. © 1994 Wiley‐Liss, Inc.
Effect of IGF‐I on pig oocyte maturation, fertilization, and early embryonic development in vitro, and on granulosa and cumulus cell biosynthetic activity
Authors: Xia Ping , Tekpetey Francis R. , Armstrong David T.
Published: February 2005

Porcine granulosa cells have been shown previously to both secrete and respond to insulin‐like growth factor‐I (IGF‐I), suggesting an autocrine function of this peptide in the follicle. The present work was undertaken to determine possible effects of IGF‐I on in vitro maturation, in vitro fertilization, and early embryonic development in culture. Granulosa and cumulus cell proliferation and differentiation based on 3H‐thymidine uptake and progesterone production, respectively, were also assessed. The results showed that the cleavage rate of oocytes was markedly stimulated in a dose‐dependent manner by the addition of IGF‐I to the oocyte maturation medium (P < 0.05). Embryo development beyond the 8‐cell stage was improved by IGF‐I, reaching a maximum of 22% at 200 ng/ml IGF‐I. Treatment with IGF‐I after fertilization increased the percentage of total oocyte cleavage (P < 0.05) to approximately 52%, 43%, and 57% at, respectively, 25, 50, and 100 ng/ml IGF‐I. 3H‐thymidine incorporation by granulosa cells was significantly increased in cultures treated with FSH (3‐fold) or IGF‐I (6‐fold) compared to the control. For the cumulus cells, FSH caused a similar increase (3‐fold) in 3H‐thymidine incorporation while IGF‐I stimulated a 15‐fold increase. Progesterone production by the granulosa cells was increased to the same extent by treatment with FSH or IGF‐I (4.7 and 5.1‐fold, respectively). However, for the cumulus cells, while FSH caused a marked 16‐fold increase in progesterone production, IGF‐I caused only a marginal increase of 2.5‐fold. These results indicate a beneficial effect of IGF‐I on in vitro porcine oocyte maturation and pre‐implantation embryo development, suggesting a physiological role for IGF‐I in vivo. The in vivo effect of IGF‐I may be indirect via autocrine stimulation of cumulus and/or granulosa cells resulting in enhanced oocyte maturation and fertilization. © 1994 Wiley‐Liss, Inc.
Biochemical characterization of the boar sperm 42 kilodalton protein tyrosine kinase: Its potential for tyrosine as well as serine phosphorylation towards microtubule‐associated protein 2 and histone H 2B
Authors: Berruti Giovanna
Published: February 2005

The majority of cellular responses to changing environmental conditions is regulated by protein kinases. Spermatozoa have many special properties, including motility with demonstrated chemotaxis, the ability to undergo capacitation, and the acrosome reaction, which are in part controlled by extracellular signals and in which sperm kinases are considered to be involved. We have previously reported that there is a protein kinase activity, which phosphorylates the synthetic substrate poly‐(Glu, Tyr) with a Km value of 2.3 μM, and is inhibited by the tyrosine kinase inhibitor tyrphostin, in the protein extract from boar spermatozoa (Berruti and Porzio, 1992: Biochim Biophys Acta 1118:149–154). Now we have demonstrated that the enzyme is cytosolic, is active as a monomer of Mr 42,000, is stimulated by Mg2+ > Mn2+ but not by Ca2+, is renaturable, and can phosphorylate native protein substrates such as microtubule‐associated protein 2 (MAP2) and histone H2B both on the tyrosine and serine residues. N‐terminal sequence analysis suggests that it is a novel protein. These new findings imply that the boar sperm 42 kD kinase may be a novel member of the emerging class of dual‐specificity protein kinases, and they raise the intriguing question of its function in the protein kinase network mediating signal transduction in mammalian spermatozoa. © 1994 Wiley‐Liss, Inc.
Modulation of spermatozoa and zona pellucida properties by the soluble acrosome reaction‐inducing factor of the ovulated egg‐cumulus complex
Authors: Boatman Dorothy E. , Magnoni Gladys E. , Robbins Robert S.
Published: February 2005

Three sources of hamster periovulatory fluids (± heat inactivation at 56°C), with bovine serum albumin (BSA) as control, were tested for effects on penetration of three classes of eggs by hamster sperm precapacitated in BSA. These fluids were a soluble extract of cumulus oophorus fluid (COF) from the ovulated hamster egg‐cumulus complex, serum, and follicular fluid. Egg types were ovulated, salt‐stored (ovulated), and follicular. In both COF and serum, there were significant differences among egg types in mean penetration, and significant effects of fluid addition. In contrast, there was no effect of follicular fluid and no differences between follicular and stored eggs. For the follicular eggs (combined data, normalized, ranked), patterns of response to the three factors (± heating) were different: only unheated COF and heated serum increased penetration significantly above BSA control levels (average rank 20.2, 41.4, 38, for BSA, COF (unheated), serum (heated), respectively). This indicated that the active component in COF was heat labile, not present in either serum or follicular fluid, and, therefore, of oviductal origin. Oviduct and/or COF exposure of eggs and sperm was tested for effects as an acrosome reaction inducing factor (ARIF) for acrosome reactions (AR; zonabound and free‐swimming sperm) and on sperm:zona binding and penetration. The COF ARIF for free‐swimming sperm AR was heat stable. Penetration of follicular eggs increased after incubation in COF prior to sperm addition, but a greater response occurred when COF was added to eggs with sperm. In kinetic experiments, 25 min following sperm attachment, follicular eggs had lost 41% of initially bound sperm, vs. 23% for ovulated eggs, and had only 16 AR sperm/egg, vs. 26 for ovulated. Follicular eggs incubated in COF (then washed three times) had the same number of bound AR sperm as ovulated eggs. Acid solubilized zona pellucida (ASZP) from ovulated eggs was more effective as an ARIF per zona than ASZP from follicular eggs. Zonae of follicular eggs, as eivdenced by dissolution times in β‐mercaptoethanol (β‐MEOH), were not “harder” than those of ovulated eggs. There were differences in lectin binding antigens on zonae of both fresh and stored, follicular and ovulated, eggs. We conclude that multiple biological factors orchestrate sperm:egg interactions in the ampulla. Our data are consistent with the presence of at least three effective components: (1) the oviductal lectin‐binding antigen (ZPO or oviductin), (2) an additional heat‐labile component, and (3) the heat‐stable ARIF for free‐swimming sperm. © 1994 Wiley‐Liss, Inc.
Mullerian‐inhibiting substance secretion is delayed in XX sex‐reversed dog embryos
Authors: Meyers‐Wallen V. N. , Palmer V. , Maclaughlin D. , Donahoe P. K.
Published: February 2005

Sertoli cell secretion of Mullerian‐inhibiting substance (MIS) begins shortly after testis differentiation. Mullerian ducts regress following MIS exposure during an embryonic critical period. In dogs with XX sex reversal, Mullerian ducts persist in the presence of testicular tissue. This study was conducted to determine whether MIS is present in ovotestes of XX sex‐reversed embryos during the period for Mullerian duct regression in normal males. XX sex‐reversed embryos and normal littermates were identified by a combination of karyotype and gonadal histology. The degree of regression in the adjacent Mullerian duct was scored. Immunohistochemical staining was used to detect MIS in the contralateral gonad. Testicular differentiation and MIS secretion were identified in XY embryos at all ages studied (35–46 days). Seminiferous tubules were not observed in gonads of embryos at risk of XX sex reversal between 35–38 days (n = 15), but were observed at 40 and 46 days (n = 3). Although positive staining for MIS was observed in ovotestes, adjacent Mullerian ducts persisted. The degree of seminiferous tubule development was reduced and MIS secretion was delayed in ovotestes, compared to normal testes. Mullerian duct persistence in this model is apparently due to an abnormality in the quantity and timing of MIS secretion during embryonic development.© 1994 Wiley‐Liss, Inc.
Role of FGFs in skeletal muscle and limb development
Authors: Olwin Bradley B. , Arthur Kirstin , Hannon Kevin , Hein Patrick , Zhou Zhaohui , Zuber Michael E. , Kudla Arthur J. , McFall Aidan , Rapraeger Alan C. , Riley Bruce , Szebenyi Györgyi , Fallon John F.
Published: February 2005

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR‐1 through FGFR‐4); (ii) a cysteine‐rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR‐1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling.

We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF‐2 or FGF‐2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF‐2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF‐2 plays an important role in limb outgrowth. We propose that FGF‐2 is an apical ectodermal ridgederived factor that participates in limb outgrowth and patterning. © 1994 Wiley‐Liss, Inc.
Role of interferons in the regulation of cell proliferation, differentiation, and development
Authors: J. Hertzog Paul , Y. Hwang Seung , Kola Ismail
Published: February 2005

There now appears to be evidence to support the view that the type I IFNs are naturally produced negative regulators of growth that also modify cell differentiation. Consistent with this, it appears that the ability to produce and respond to IFN is suppressed in early embryonic development when cell proliferation and differentiation are essential. In the later stages of fetal development, IFN production is de‐repressed, and cells show increased sensitivity to IFN, which may be important in regulating cell proliferation and/or differentiation processes or the interaction between fetal and maternal tissues. Interestingly, the IFN system can also be suppressed in disease states such as the development of tumours or in the establishment of a (chronic) viral infection. Therefore, understanding the developmental regulation of the IFN system may be important to understanding and controlling the IFN system in disease. More extensive studies of the developmental stage and tissue‐specific expression of type I IFNs and their receptors are necessary, as well as more direct in vivo experiments to further elucidate the role of the IFN system in reproduction and development. © 1994 Wiley‐Liss, Inc.
Affinity binding of hamster oviductin to spermatozoa and its influence on in vitro fertilization
Authors: Kimura Hitomi , Matsuda Junichiro , Ogura Atsuo , Asano Toshihiko , Naiki Masaharu
Published: February 2005

The aim of this study was to determine the influence of hamster oviductal glycoprotein (Oviductin) on in vitro gamete interaction. Oviductin was purified from the oviducts using lithium 3,5‐diiodosalicylate, followed by phenol extraction. Immunocytochemistry using indirect fluorescence staining revealed that oviductin binds to the sperm anterior acrosomal region. The specific binding of oviductin resulted in inhibition of in vitro fertilization in studies using cumulus‐free oocytes. The inhibitory effect was dependent on the concentration of oviductin and occurred in both ovarian and oviductal oocytes but not zona‐free oocytes, indicating that sperm‐zona interaction was interferred by oviduction. However, the inhibitory effect of oviductin sperm‐zona interaction was reduced when cumulus‐enclosed oocytes from ovaries and oviducts were used, indicating that the egg investment including cumulus oophorus has some effect on oviductin‐sperm complex and maintaining the fertilizing ability. © Wiley‐Liss, Inc.
Analysis of aspartic acid and asparagine metabolism in Xenopus laevis oocytes using a simple and sensitive HPLC method
Authors: M. O'Connor Clare
Published: February 2005

The amino acids in methanol‐soluble extracts of Xenopus oocytes were measured using a method involving precolumn derivatization with phenylisothiocyanate and reverse phase HPLC of the derivatized amino acids. This technique allows the estimation of asparagine and glutamine pools in oocytes, estimated as 70 and 283 pmoles per oocyte, respectively. The pool sizes of the other amino acids were similar to previously reported results obtained using conventional ion exchange chromatography and postcolumn derivatization with ninhydrin. The advantages of the method developed here include picomolar sensitivity and the enhanced resolution of asparagine and glutamine from other amino acids. The kinetics of aspartic acid and asparagine utilization were monitored following microinjection of oocytes with [3H]aspartic acid and [14C]asparagine. The aspartic acid pool turned over rapidly with a half‐time of <30 min. The asparagine pool was metabolized much more slowly and appeared to be utilized almost completely for protein synthesis. The absolute rate of protein synthesis in oocytes was calculated from the incorporation data and chemical pool measurements as ∼25 ng/hr‐oocyte. The methodology developed here may be useful in experimental situations involving limited amounts of biological material. © 1994 Wiley‐Liss, Inc.