bluuebunny/crossref_metadata_2025 huggingface dataset:
Alterations in development of reproductive and endocrine systems of wildlife populations exposed to endocrine-disrupting contaminants
Authors: Guillette LJ  , Gunderson MP
Published: March 2004

Wildlife and human populations are affected by contaminants in natural settings. This problem has been a growing concern over the last decade with the realization that various environmental chemicals can alter the development and functioning of endocrine organs, cells and target tissues. Documented disruptions or alterations in reproductive activity, morphology or physiology in wildlife populations have been correlated with contaminant-induced modifications in endocrine system functioning. Alterations of the endocrine system are complex, and not limited to a particular organ or molecular mechanism. For instance, contaminants have been shown to (1) act as hormone receptor agonists or antagonists, (2) alter hormone production at its endocrine source, (3) alter the release of stimulatory or inhibitory hormones from the pituitary or hypothalamus, (4) alter hepatic enzymatic biotransformation of hormones, and (5) alter the concentration or functioning of serum-binding proteins, altering free hormone concentrations in the serum. This review focuses on two of these alterations, altered hormone synthesis and hepatic biotransformation, as a number of recent studies indicate that these actions are important components of endocrine disruption in developing organisms. The possible role of contaminants in altering sex determination mechanisms is also examined.
Expression of vascular endothelial growth factor (VEGF) receptors in rat corpus luteum: regulation by oestradiol during mid-pregnancy
Authors: Sugino N , Kashida S , Takiguchi S , Karube-Harada A , Kato H
Published: March 2004

The aim of this study was to investigate the expression of vascular endothelial growth factor (VEGF) receptors, the fms-like tyrosine kinase (flt-1) and kinase insert domain-containing region (KDR), in corpora lutea obtained at different stages of the oestrous cycle and during pregnancy in rats. Immunohistochemistry revealed that both flt-1 and KDR were localized in luteal cells in addition to vascular endothelial cells, and that the intensity of staining was stronger in pregnant rats than in cyclic rats. Rats undergoing hypophysectomy-hysterectomy on day 12 of pregnancy were treated with oestradiol until day 15 of pregnancy to determine whether oestradiol is involved in expression of flt-1 and KDR mRNA in the corpus luteum during mid-pregnancy. The flt-1 and KDR mRNA contents in the corpus luteum were decreased significantly by hypophysectomy-hysterectomy, and these decreases recovered significantly after oestradiol treatment. Changes in the mass of the corpus luteum and serum progesterone concentrations paralleled the changes in expression of flt-1 and KDR mRNA. Developmental studies indicated that flt-1 and KDR mRNA contents in the corpus luteum were constant until day 15 of pregnancy but decreased significantly on day 21 of pregnancy. In conclusion, both flt-1 and KDR were expressed in luteal cells in addition to vascular endothelial cells, and expression was upregulated by oestradiol during mid-pregnancy. flt-1 and KDR may play a role in development of the corpus luteum and in production of progesterone during mid-pregnancy in rats.
Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation
Authors: Green CE , Watson PF
Published: March 2004

Cryopreserved spermatozoa demonstrate reduced conception rates compared with fresh spermatozoa when used for artificial insemination. The preliminary stage of cryopreservation of spermatozoa involves cooling to 5 degrees C, during which spermatozoa experience a capacitation-like change, which may be partially responsible for the reduced conception rate observed. The aim of this study was to determine the nature of these capacitation-like changes and how much this process resembles true capacitation. Boar spermatozoa, cooled to 5 degrees C and re-warmed to physiological temperatures (39 degrees C), were compared with spermatozoa capacitated in Tyrode's complete medium (TALP) for 2 h at 39 degrees C. Fluorescent probes, and SDS-PAGE and western blotting were used to visualize events known to occur during capacitation in vitro. Chlortetracycline staining of membrane domains and Fluo-3 detection of changes in intracellular free calcium by flow cytometry in cooled and re-warmed spermatozoa showed similarities to those of capacitated spermatozoa. Alterations to lipid bilayer fluidity assessed by merocyanine fluorescence staining and intracellular signalling pathways detected by tyrosine phosphorylation of cooled and re-warmed spermatozoa, did not completely reflect the changes detected during capacitation in vitro. Thus, cooling spermatozoa to 5 degrees C results in a similar endpoint to that observed in capacitated cells in terms of reactive membranes and changes in intracellular ion concentrations, which may account for their comparable functionality. However, these modifications are not completely analogous and should not be considered true capacitation, but rather a by-passing of the capacitation process.
Relaxin-like factor (RLF) serum concentrations and gubernaculum RLF receptor display in relation to pre- and neonatal development of rats
Authors: Boockfor FR , Fullbright G , Bullesbach EE , Schwabe C
Published: March 2004

Deletion of the relaxin-like factor (RLF) gene in mice causes retention of testicles and infertility. The development of a synthetic RLF has made it possible to investigate the events that connect the genomic event and the basic biological responses that cause gonadal positioning. Anti-RLF antibodies were raised against synthetic RLF, allowing determination of RLF concentrations during the critical period, testing for RLF receptors on the gubernaculum and exploration of the temporal relationship between receptor display and migration of the testes in developing rats. In male rat pups, serum RLF concentrations were high at day 2 before parturition (2.4 ng ml(-1)) and decreased sharply just before parturition. Thereafter, males and females had the same low serum concentrations until RLF concen-trations began to increase in males only, starting at day 10 after parturition and continuing until adult RLF concentrations (0.6 ng ml(-1)) were reached on day 39 after parturition. The testicles are descending into the scrotum during this phase of increasing RLF concentrations and are descended fully by day 19-21 after parturition, before adult hormone concentrations are established. The high prenatal serum RLF concentration coincides with high expression of RLF receptors in the gubernaculum tissue. Competitive binding of RLF per mg of membrane protein prepared from rat gubernacula at various developmental stages showed no increase in receptor density as sexual maturity was reached. Gubernaculum cells in primary culture showed an increased uptake of 5-bromo-2'-deoxyuridine in the presence of RLF compared with controls. These studies demonstrate that the synthetic RLF is biologically active and indicate that the cryptorchid phenotype INSL3(-/-) is a direct consequence of defective gubernaculum growth, caused by the absence of RLF during early phases of development.
Increased expression of major histocompatibility complex (MHC) class I transplantation antigens in bovine trophoblast cells before fusion with maternal cells
Authors: Bainbridge DR , Sargent IL , Ellis SA
Published: March 2004

The mammalian fetus is potentially at risk from maternal immune attack because it can express paternally inherited polymorphic antigens, including those encoded by the major histocompatibility complex (MHC). The aim of this study was to investigate in more detail MHC class I upregulation by binucleate trophoblast cells in the bovine placenta. A method was developed to isolate binucleate cells by enzymatic disaggregation and density gradient centrifugation of bovine placental cotyledons. In cytospin preparations, 25-30% of purified binucleate cells stained positively with antibodies that recognize bovine MHC class I. The same antibodies were used to immunoprecipitate radiolabelled class I molecules from lysates of binucleate cells and fetal peripheral blood mononuclear cells. The protein species isolated from the two types of cell were similar in size and degree of glycosylation. PCR amplification of cDNA generated from binucleate cells and subsequent sequence analysis demonstrated transcription of MHC class I mRNA species similar to those found in fetal peripheral blood mononuclear cells, and there was no evidence of genetic imprinting of paternally inherited alleles. These results indicate that binucleate cells upregulate expression of MHC class I as they differentiate from MHC-negative uninucleate trophoblast cells. This finding has important implications for the immunological status of the fetus, as binucleate trophoblast cells are destined to cross to the maternal side of the placenta where they fuse with maternal cells. The immunological function of the resulting antigenically mixed fetomaternal hybrid minisyncytia is unknown.
Effect of maternal undernutrition during pregnancy on early ovarian development and subsequent follicular development in sheep fetuses
Authors: Rae MT , Palassio S , Kyle CE , Brooks AN , Lea RG , Miller DW , Rhind SM
Published: March 2004

Gonad development in female sheep fetuses is thought to occur in a number of key stages. The aim of this study was to determine the effects of maternal undernutrition, applied at one or more of these critical stages, on fetal ovarian development. Groups of ewes (n = 11-19) were fed rations providing either 100% (high; H) or 50% (low; L) of energy requirements for live weight maintenance during selected 'windows' during gestation. Control ewes (HH and HHH) were fed the H ration from mating until they were killed at days 50, 65 (HH) or 110 (HHH) of gestation, whereas ewes of other groups were fed the L ration for the periods between day 0 and day 30 of gestation (LH and LHH), day 31 and day 50 or 65 of gestation (HL and HLH), day 65 and day 110 of gestation (HHL) or day 0 of gestation until the animals were killed (LL and LLL). At day 50 of gestation, there was no effect of nutritional treatment on mean fetal mass but compared with HH animals, mean fetal ovarian mass was significantly lower in HL (P < 0.05) and LL (P < 0.001) animals. At day 65 of gestation, there were significantly fewer germ cells (P < 0.05) at the resting, diplotene stage of initial meiosis in LL animals than there were in HH animals, indicating delayed germ cell maturation and onset of meiosis. Qualitative assessment of proliferative cell nuclear antigen immunostaining indicated that, at day 50 of gestation, staining was located predominantly in the germ cells, whereas by day 65 of gestation, staining was confined predominantly to somatic cells. Undernutrition in each one of these windows was associated with delayed ovarian follicular development (P < 0.05-0.001) as measured by development of the granulosa cell layer at day 110 of gestation. This study demonstrates that undernutrition before and during folliculogenesis can delay fetal follicular development.
Expression of uterine and cervical epithelial cadherin during relaxin-induced growth in pigs
Authors: Ryan PL , Baum DL , Lenhart JA , Ohleth KM , Bagnell CA
Published: March 2004

Epithelial cadherin (E-cadherin), a member of the cadherin family of calcium-dependent adhesion molecules, is present in reproductive tissues. Relaxin, a hormone important for uterine and cervical growth in pigs, increases the expression of E-cadherin in the MCF-7 mammary epithelial cell line. The objective of this study was to characterize the expression of E-cadherin during relaxin-induced growth of the uterus and cervix in an immature pig model, independent of high circulating steroids. After administration of relaxin to prepubertal gilts, the uterus and cervix were collected. E-cadherin mRNA and protein were measured by northern and western blot analysis, respectively. A 120 kDa protein band, corresponding to E-cadherin, was detected in all tissues examined. Relaxin significantly (P < 0.05) increased the amount of E-cadherin protein in the uterus (P < 0.05), whereas no significant changes were observed in E-cadherin protein in the cervix. A 4.2 kb E-cadherin transcript was detected in all tissues and E-cadherin mRNA was significantly higher (P < 0.05) in uteri from relaxin-treated gilts compared with control gilts. E-cadherin was localized by immunocytochemistry to the epithelial cells of the uterine and cervical lumen, and the uterine glandular epithelium. Quantitative analysis revealed that administration of relaxin significantly increased (P < 0.05) the height of the uterine luminal epithelium compared with that of the controls. This is the first report of the expression of E-cadherin in the uterus and cervix of pigs. The findings from this study indicate that relaxin increases the expression of uterine E-cadherin in the reproductive tract of pigs. Administration of relaxin to prepubertal gilts in vivo increased uterine epithelial cell growth independent of circulating steroids, with a concomitant increase in E-cadherin expression.
Expression of oxytocin, oestrogen and progesterone receptors in uterine biopsy samples throughout the oestrous cycle and early pregnancy in cows
Authors: Robinson RS , Mann GE , Lamming GE , Wathes DC
Published: March 2004

This study examined the expression patterns of oxytocin and steroid receptors in the bovine endometrium during the oestrous cycle and early pregnancy to elucidate their respective roles in the regulation of luteolysis and the maternal recognition of pregnancy. In Expt 1, uterine biopsies were collected from four cows throughout three oestrous cycles each, to provide daily samples. In Expt 2, uterine tissue was collected on days 12, 14, 16 and 18 of the oestrous cycle (n = 20) or early pregnancy (n = 16). Oxytocin receptor, oestrogen receptor alpha and progesterone receptor mRNAs were localized by in situ hybridization, and localization of oestrogen receptor and progesterone receptor was confirmed by immunocytochemistry. All three receptors showed time- and cell-specific expression patterns. Oestrogen receptor alpha increased in all regions at oestrus but high concentrations were also found in the luminal epithelium during the mid-luteal phase and in the deep glands throughout the oestrous cycle. Progesterone receptor expression was higher in the stroma than it was in the types of epithelial cell, and increased expression was observed at oestrus and during the early luteal phase. The cyclical upregulation of oxytocin receptors in the luminal epithelium on about day 16 was not related to preceding changes in the endometrial expression of either oestradiol alpha or progesterone receptors. During early pregnancy, oxytocin receptor expression was suppressed. Oestrogen receptor a concentrations increased in the non-pregnant cows and decreased in the pregnant cows between days 16 and 18, but these changes followed rather than preceded the upregulation of oxytocin receptors in the non-pregnant cows. It is concluded that the initial upregulation of oxytocin receptors in the luminal epithelium, which triggers luteolysis, is not associated directly with changes in expression of oestrogen receptor alpha.
Gap-junctional communication in mouse cumulus-oocyte complexes: implications for the mechanism of meiotic maturation
Authors: Webb RJ , Bains H , Cruttwell C , Carroll J
Published: March 2004

The mechanisms underlying the hormonal stimulation of meiotic maturation are not understood. The most prevalent hypothesis is that hormone-induced maturation is stimulated by an increase in the intracellular messengers, cAMP or Ca2+. This study investigated whether Ca2+ transients in somatic cells can lead to Ca2+ transients in the oocyte, and whether hormones that stimulate meiotic maturation of mouse oocytes in vitro and in vivo stimulate an increase in intracellular Ca2+. Of a range of potential agonists of Ca2+ release, ATP and UTP were the only agents that stimulated Ca2+ release in cumulus cells. ATP-induced Ca2+ release is from intracellular stores, as the response is not blocked by chelation of extracellular Ca2+, but is inhibited by the Ca2+-ATPase inhibitor, thapsigargin. ATP and UTP are equipotent, consistent with the receptor being of the P2Y2 type. Confocal microscopy was used to show that ATP-induced Ca2+ release in cumulus cells leads to a Ca2+ increase in the oocyte. Inhibition of gap-junctional communication using carbenoxolone, as assayed by dye transfer, inhibited the diffusion of the Ca2+ signal from the cumulus cells to the oocyte. Thus, provided that a Ca2+ signal is generated in the somatic cells in response to maturation-inducing hormones, it is feasible that a Ca2+ transient is generated in the oocyte. However, FSH and EGF, both of which stimulate maturation in vitro, have no effect on Ca2+ in cumulus--oocyte complexes. Furthermore, LH, which leads to meiotic maturation in vivo, did not stimulate Ca2+ release in acutely isolated granulosa cells from preovulatory mouse follicles. These studies indicate that ATP may play a role in modulating ovarian function and that diffusion of Ca2+ signals through gap junctions may provide a means of communication between the somatic and germ cells of the ovarian follicle. However, our data are not consistent with a role for Ca2+-mediated communication in hormone-mediated induction of meiosis in mice.
Effects of ovarian follicle ablation on FSH, oestradiol and inhibin A concentrations and growth of other follicles in sheep
Authors: Evans AC , Flynn JD , Duffy P , Knight PG , Boland MP
Published: March 2004

The aim of this study was to examine the effect of removal of the largest follicle or all visible follicles during the first follicle wave on subsequent follicular growth, steroid, inhibin A and gonadotrophin secretion in sheep. On day 4.5 of a synchronized oestrous cycle, ewes (n = 18) were assigned to one of three groups which underwent either no treatment (control), ablation of the largest follicle (largest follicle aspirated and cauterized via laparotomy) or ablation of all follicles (all visible follicles ablated). Between day 0 and day 10 of the oestrous cycle, blood samples were collected every 8 h and ovaries were examined daily using transrectal ultrasonography. The lifespan of the second largest follicle (number of days > 3 mm in diameter) was longer (6.7 +/- 0.9 days; P < 0.05) and the maximum diameter tended to be greater (4.8 +/- 0.3 mm; P = 0.07) in ewes in which the largest follicle was ablated than in the control ewes (3.8 +/- 0.4 days; 4.2 +/- 0.3 mm). There was no difference in the day of emergence of the second follicular wave between groups (day 6.9 +/- 0.4). However, the peak of the transient increase in FSH concentrations after ablation was earlier (day 5.67 +/- 0.15; P < 0.05) in ewes in which all follicles were ablated than in control ewes (day 6.72 +/- 0.36); the timing in ewes that had only the largest follicle ablated was intermediate (day 6.11 +/- 0.28). Serum inhibin A concentrations were about three-fold lower (P < 0.05) in both follicle ablation groups than in the control group. The numbers of follicles 2-3 mm in diameter during the first 3 days of the second follicular wave were greater in 'ablated ewes' (both groups had 2.6 +/- 0.2 follicles day-1) than in control ewes (1.7 +/- 0.3 follicles day-1). It is concluded that: (i) transient increases in FSH concentrations precede the emergence of follicle waves; (ii) ablation of all follicles on day 4.5 after oestrus advanced the timing of the next peak in FSH concentrations and the numbers of small follicles associated with the development of the second follicular wave; and (iii) ablation of the largest follicle resulted in an increase in the lifespan of the second largest follicle, indicating a regulatory role of large dominant follicles over smaller subordinate follicles.
Segregation of spermatozoa within sperm storage tubules of fowl and turkey hens
Authors: King LM , Brillard JP , Garrett WM , Bakst MR , Donoghue AM
Published: March 2004

In avian species, spermatozoa reside in the oviduct for prolonged periods in specialized structures known as sperm storage tubules, but little is known about the relative distribution of spermatozoa in these tubules after successive inseminations by different males. The staining efficacies of various fluorescent dyes for fowl and turkey spermatozoa were evaluated to investigate one proposed mechanism of sperm competition. Hens were then inseminated at different intervals with stained and unstained spermatozoa to observe the spatial distribution of spermatozoa within the storage tubules. Several novel fluorescent lipophilic tracers that successfully stain mammalian spermatozoa either did not stain fowl or turkey spermatozoa, or greatly impaired sperm motility. In contrast, Hoechst 33342 readily stained sperm nuclei (fowl: 25 nmol l-1; turkey: 77 nmol l-1) within 4 h without inhibiting sperm motility, or affecting fertility or the hatching ability of the eggs. Hens were tandemly inseminated with equal numbers of stained or unstained spermatozoa at 24 h intervals and were killed 24 h after the final insemination to study sperm entry and storage within the tubules. Oviductal mucosa containing sperm storage tubules was removed, and individual tubules were classified as containing stained spermatozoa, unstained spermatozoa, a mixture of stained and unstained spermatozoa, or as not containing spermatozoa. Results from the present study indicate that spermatozoa from two different inseminations generally segregate into different storage tubules in both fowl and turkey hens. Storage tubules containing mixed populations of spermatozoa were found in only 4% of fowl and 12% of turkey storage tubules examined. Thus, the mechanism of last-male precedence does not appear to be due to the stratification of spermatozoa within the tubules.
Effects of ovarian steroids and epidermal growth factor (EGF) on expression and bioactivation of specific regulators of transcription and translation in oviductal tissue in pigs
Authors: Wollenhaupt K , Tomek W , Brussow KP , Tiemann U , Viergutz T , Schneider F , Nurnberg G
Published: March 2004

The epidermal growth factor receptor (EGF receptor) system is involved in regulation of proliferation and differentiation in oviductal and endometrial tissues. In this study the influence of ovarian steroids and EGF on the expression and activity of specific markers of transcription (mitogen-activated protein kinase; MAP42k) and translation (a potential repressor of eukaryotic initiation factor 4E; 4E-BP1) in pig oviducts was investigated. Furthermore, determination of the distribution of translationally active (polysomal) and repressed (free) mRNA, and cell cycle analysis were performed. Oviductal tissue collected at two points of the oestrous cycle (days 12 and 20) from gilts and tissues from ovariectomized gilts with or without steroid replacement treatment were analysed. The influence of EGF was detected by culture of oviductal explants. MAP42k activity was stimulated by oestrogen treatment, whereas progesterone treatment appeared to decrease its activity. High oestrogen but not high progesterone concentrations resulted in reduced mobility of 4E-BP1 on polyacrylamide gels, indicating its inactivation. EGF and oestrogen treatment of oviductal explants further reduced the mobility of 4E-BP1 on polyacrylamide gels. High concentrations of oestrogen in the plasma promoted cell cycle activity. Progesterone treatment alone did not stimulate the rate of DNA synthesis. There were no significant differences in the distribution of free oviductal poly (A+) mRNA, but the amount of polysomal mRNA was downregulated by oestrogen and progesterone. Increased oestrogen concentrations are involved in the regulation of MAP42k and 4E-BP1 activation in the oviductal tissue of pigs. The effect of oestrogen and EGF in reducing the mobility of 4E-BP1 on gels in oviductal explants indicates that EGF may mediate the effect of oestradiol in the oviducts.
Comparison of uteroplacental glycosylation in the camel (Camelus dromedarius) and alpaca (Lama pacos)
Authors: Jones CJ , Abd-Elnaeim M , Bevilacqua E , Oliveira LV , Leiser R
Published: March 2004

The recent birth of a camel-llama hybrid, after numerous failed attempts, has prompted an investigation into the glycosylation of apposing fetal and maternal tissues of pregnant camels and alpacas. This study was undertaken to determine whether interspecies differences in glycans are factors that may account in part for the difficulty in producing a viable hybrid. Specimens of camel placentae from day 60 to day 375 of gestation and alpaca placentae from day 22 to term (approximately 345 days) were fixed and embedded in resin, and sections were stained with a panel of 19 biotinylated lectins and an avidin--peroxidase revealing system. Several qualitative interspecies differences in tissue glycosylation were found, mainly in the trophoblast, and especially with respect to bi/tri-antennary bisected N-glycan, fucosylated structures, beta-galactosyl residues and sialyl termini. In the maternal uterine epithelium, differences were found mainly in bi/tri-antennary bisected complex N-glycan and beta-galactosyl residues, indicating that there is more conservation of glycosylation in maternal tissues compared with trophoblast. There were also many quantitative differences in the distribution of glycans. It is possible that a failure to effect the normal glycan--glycan complementation that occurs at the cell surface between maternal and fetal tissues during the implantation processes of apposition and adhesion may account in part for the difficulty in establishing a viable pregnancy between these two species.
Expression of luteinizing hormone genes in bovine conceptuses
Authors: Otieno CJ , Wood CM , Tallam SK , Verrinder Gibbins AM , Walton JS
Published: March 2004

RT-PCR analysis demonstrated that bovine conceptuses at days 16, 23 and 30 expressed LH-beta-like and glycoprotein hormone alpha-like transcript sequences; adult kidney, liver and brain produced predominantly unspliced products. Sequencing of the LH-beta-like fragment (from conceptuses at day 30) indicated complete homology with the published sequence. In addition, ribonuclease protection assay of RNA samples from bovine conceptuses at day 30 with a bovine LH-beta probe revealed the presence of protected molecules that appeared to be full length. Northern blot analysis of total RNA from conceptuses at day 30 failed to demonstrate the presence of LH-beta or glycoprotein alpha subunit transcripts, whereas both transcripts were readily detected in adult pituitary RNA. Administration of hCG into the uterus of heifers from day 14 to day 16 of the oestrous cycle did not affect circulating progesterone concentrations, whereas the same dose increased progesterone concentrations (P < 0.05) when administered intravenously. These results indicate that the early bovine conceptus transcribes genes encoding LH-alpha and -beta subunits, but at a level unlikely to be of physiological consequence.
Voltage-operated calcium channels in male germ cells
Authors: Jagannathan S , Publicover SJ , Barratt CL
Published: March 2004

The acrosome reaction is a key event in fertilization. Current models for induction of the acrosome reaction incorporate a necessary influx of Ca(2+), which is mediated by agonist-induced gating of ion channels in the sperm plasma membrane. The difficulty of applying electrophysiological techniques to spermatozoa has severely hampered studies on the expression of functional ion channels in these cells. However, during the last few years, a combination of molecular and physiological techniques (applied to immature spermatogenic cells) has elucidated both the expression of Ca(2+) channels in male germ cells and their role in induction of the acrosome reaction. It now appears that a range of voltage-operated Ca(2+) channels, similar to those that occur in somatic cells, is expressed in spermatozoa. Male rodent germ cells express a low-voltage activated (T-type) channel that is regulated by membrane potential and provides the primary Ca(2+) influx mechanism in zona pellucida-stimulated spermatozoa. In human spermatozoa, similar channels are apparently expressed, but their function in induction of the acrosome reaction has yet to be established. A range of other, high voltage-activated channels also appear to be present in rodent and human spermatozoa, but their roles are not yet known. In this review, the structure and characteristics of voltage-operated Ca(2+) channels are outlined and the evidence for their expression and function in male germ cells is assembled and discussed.
Expression of a novel reticulon-like gene in human testis
Authors: Zhou ZM , Sha JH , Li JM , Lin M , Zhu H , Zhou YD , Wang LR , Wang YQ , Zhou KY
Published: March 2004

Identification of genes that are specifically expressed in the adult testis or the fetal testis is important for the study of genes related to the development of the testis. In this study, a human testis cDNA microarray was established. PCR products of 9216 clones from a human testis cDNA library were dotted on a nylon membrane; mRNA from adult and fetal testes were purified and probes were prepared by a reverse transcription reaction with testis mRNA as template. The microarray was hybridized with probes of adult and fetal testes, and 96.8 and 95.4% of clones were positive, respectively. In total, 731 clones were differentially expressed: 592 were highly expressed in adult testis and 139 were highly expressed in fetal testis. Among these genes, a new reticulon (Rtn)-like gene was detected and named Rtn-T. Rtn-T was highly expressed in adult human testis. The cDNA of Rtn-T contains 3491 bp and the putative protein had 968 amino acids. This protein is homologous to the six known members of the Rtn family (KIAA0886, Rtn xL, reticulon 4a, Nogo-A, Nogo-A short form, and brain my043) but was different at the 5' end. All homologues originate from one gene, and result from both different promotor regions and different splicing. Rtn-T lacks the first exon and contains a second exon that is lacking in the other homologues. Rtn-T is shorter than KIAA0886, Rtn xL, reticulon 4a and Nogo-A, but longer than the Nogo-A short form and brain my043. Sequence analysis showed that Rtn-T protein has two hydrophobic regions that may be membrane-spanning domains. Expression profiles showed that Rtn-T is specifically and strongly expressed in testis. The results of the present study indicate that the Rtn-T gene is differentially expressed in adult and fetal testes and encodes a membrane protein that may have a function in testis development.
Use of energy substrates by various stage preimplantation pig embryos produced in vivo and in vitro
Authors: Swain JE , Bormann CL , Clark SG , Walters EM , Wheeler MB , Krisher RL
Published: March 2004

The aim of in vitro embryo systems is to produce embryos of comparable quality to those derived in vivo. Comparison of embryo metabolism as an indicator of viability may be useful in optimization of culture conditions. The aim of the present study was to determine glucose, glutamine and pyruvate use by various stage pig embryos produced in vitro and in vivo. The results indicate that pig embryos use glucose via glycolysis in significant amounts at all stages examined, regardless of embryo origin. In vitro-derived embryos have significantly increased glycolytic activity after the eight-cell stage, whereas in vivo-derived embryos have increased glycolysis at the blastocyst stage. In vivo-derived embryos have higher rates of glycolysis compared with in vitro-derived embryos. Glucose usage through the Krebs cycle for in vitro- and in vivo-derived embryos increased significantly at the blastocyst stage. Pig embryos produced in vitro used constant amounts of glutamine throughout development, whereas in vivo-derived embryos increased glutamine usage after the eight-cell stage. Pyruvate use was minimal at all stages examined for both in vitro- and in vivo-derived pig embryos, showing significant increases at the blastocyst stage. Krebs cycle metabolism of pyruvate, glutamine and glucose by in vivo-derived embryos was higher than that by in vitro-derived embryos. Current in vitro culture conditions produce pig embryos with altered metabolic activity, which may compromise embryo viability.
Modulation of sex hormone secretion in cows by acute infection with bovine viral diarrhoea virus
Authors: Fray MD , Mann GE , Bleach EC , Knight PG , Clarke MC , Charleston B
Published: March 2004

Bovine viral diarrhoea virus (BVDV) is a major pathogen of cattle and is responsible for considerable reproductive loss. In this study, the in vivo responses in six multiparous cows were investigated after a non-cytopathogenic BVDV challenge (strain Pe 515; 5 x 10(6) tissue culture infective dose 50) given 9 days before a synchronized ovulation. Six similar cows challenged with non-infectious culture medium served as controls. The experimental noncytopathogenic BVDV infection was followed by a viraemia and leucopenia at days 5-9 after challenge, but no other clinical signs of infection were detected. However, the BVDV infection altered endocrine function. Mean LH pulse frequency immediately before CIDR withdrawal was lower (P < or = 0.05) in the BVDV-infected (2.17 +/- 0.34 pulses per 8 h) compared with the sham-infected (4.83 +/- 1.04 pulses per 8 h) animals. At day 3 after CIDR withdrawal, plasma oestradiol concentrations remained high (P < 0.05) in the infected cows (2.19 +/- 0.51 pg ml(-1)) compared with the sham-infected controls (0.72 +/- 0.29 pg ml(-1)). However, there was no difference in the peak oestradiol concentration (BVDV: 2.31 +/- 0.29 versus sham: 2.34 +/- 0.41 pg ml(-1)). In addition, non-cytopathogenic BVDV significantly (P < 0.05) increased the duration of the interval between ovulation and onset of the postovulatory progesterone increase (values 1.0 ng ml(-1)) (BVDV: 3.0 +/- 0.26 versus sham: 4.0 +/- 0.26 days). The viral infection also significantly (P < 0.01) decreased mean plasma progesterone concentrations between day 3 and day 11 after ovulation (BVDV: 2.59 +/- 0.32 versus sham: 4.13 +/- 0.27 ng ml(-1)). These data show that non-cytopathogenic BVDV viraemias during the follicular phase can modulate the secretion of gonadotrophins and sex steroids, in particular progesterone, during a synchronized oestrous cycle. Therefore, viraemias during the follicular phase may reduce the fertility of cattle by disrupting the capacity of the ovulatory follicle to form a competent corpus luteum, thereby compromising early embryo development and maternal recognition of pregnancy.
Modulatory effects of gonadotrophins and insulin-like growth factor on the secretion of inhibin A and progesterone by granulosa cells from chicken preovulatory (F1-F3) follicles
Authors: Lovell TM , Gladwell RT , Groome NP , Knight PG
Published: March 2004

The aim of this study was to compare the actions and interactions of gonadotrophins (LH and FSH) and an analogue of insulin-like growth factor I (LR3-IGF-I) on the secretion of inhibin A, inhibin B and progesterone by cultured chicken granulosa cells derived from the three largest (F1--F3) follicles of the preovulatory hierarchy. Treatment with LH or FSH promoted marked dose-(P < 0.0001) and time- (P < 0.0001) dependent increases in both inhibin A and progesterone secretion, with the magnitude of response (< 15-fold compared with basal) increasing over time in culture. Concentrations of inhibin B were below the detection limit in all samples. Initially, F1 cells were more LH-responsive than were F3 cells in terms of progesterone secretion (P < 0.02) but this difference between follicles decreased over time in culture. In contrast, LH-induced inhibin A secretion tended to be highest from F3 cells, although this was not significant. Cells from F3 follicles were consistently more FSH-responsive than F1 cells in terms of both progesterone (P < 0.01) and inhibin A (P < 0.02) secretion. Initially, F1 cells were more responsive to LR3-IGF-I than were F3 cells in terms of progesterone secretion (P < 0.001) but were less responsive in terms of inhibin A secretion (P < 0.001). Again, these inter-follicle differences decreased over time in culture (not significant on day 3 of treatment). Co-treatment experiments showed that LR3-IGF-I enhanced both LH- and FSH-induced secretion of inhibin A and progesterone in a time- (P < 0.001) and follicle- (P < 0.001) dependent way. Initially, F1 cells showed highest LR3-IGF-I enhancement of LH-induced inhibin A and progesterone secretion; in contrast, F3 cells showed the highest LR3- IGF-1 enhancement of FSH-induced inhibin A and progesterone secretion. These inter-follicle differences persisted over time in the case of FSH-induced hormone responses but not in the case of LH-induced responses, even though the relative degree of LR3-IGF-I enhancement increased markedly over time. Collectively, these data support a positive role for IGF-I, presumably of thecal origin, as an amplifier of gonadotrophin action on granulosa cell inhibin A and progesterone production by preovulatory chicken follicles.
Induction of birth in the bandicoot (Isoodon macrourus) with prostaglandin and oxytocin
Authors: Gemmell RT
Published: March 2004

As in eutherians, maturation of the fetal pituitary and adrenal glands together with an increase in prostaglandin and mesotocin or oxytocin production initiates birth in marsupials. In this study, prostaglandin (Lutalyse) or oxytocin (Syntocinon) were administered to pregnant bandicoots at 05:00 h on the calculated day of birth and the resultant effects were filmed for analysis. The administration of prostaglandin caused the bandicoot to adopt the birth position several minutes after injection (n = 2). However, the bandicoot did not give birth for several hours. Birth occurred at a similar time of day to that observed for untreated bandicoots (n = 7), between 08:00 h and 12:00 h. After an injection of oxytocin, the bandicoot assumed the birth position and birth occurred within several minutes. The young were alive while still connected to their allantoic stalks. However, they were unable to attach to the teats and did not survive (n = 4). The induced young were the colour of venous blood and died soon after the umbilicus was separated, indicating that the cardiopulmonary system of these neonates was underdeveloped and inadequate to maintain life. The results from this study demonstrate that prostaglandin is required to prepare the bandicoot for birth, and mesotocin is required for contraction of the uterus and for birth to occur.
Importance of cooling rate and animal variability for boar sperm cryopreservation: insights from the cryomicroscope
Authors: Medrano A , Watson PF , Holt WV
Published: March 2004

A series of experiments was set up to investigate the effect of different cooling rates on boar sperm cryosurvival using cryomicroscopy. The cooling protocols were split into two stages: (i) from +5 degrees C to -5 degrees C and (ii) from -5 degrees C to -50 degrees C. Fluorescent probes (SYBR14 and propidium iodide) were used to monitor plasma membrane integrity during the entire process. Cooling rates in the range 3 degrees C min(-1) to 12 degrees C min(-1) did not cause significant damage to the sperm plasma membrane between +5 degrees C and -5 degrees C; however, spermatozoa cooled at 24 degrees C min(-1) to -5 degrees C were slightly damaged. Motility was not particularly sensitive to variations in cooling rate. Cooling rates in the range 15 degrees C min(-1) to 60 degrees C min(-1) did not produce differences in sperm cryosurvival during freezing between -5 degrees C and -50 degrees C, or after thawing. In addition, cooling rates in the range 3 degrees C min(-1) to 80 degrees C min(-1) did not produce significant differences in sperm cryosurvival. However, slow freezing (3 degrees C min(-1)) induced a slight increase in the percentage of plasma membrane-damaged spermatozoa (propidium iodide-positive) at -50 degrees C. Inter-ejaculate and inter-boar differences in sperm cryosurvival were manifested independently of cooling rate. The sperm plasma membrane remained intact (SYBR14-positive) during cooling and freezing, but upon rewarming, the plasma membrane of a high proportion of spermatozoa was damaged (propidium iodide-positive), indicating that rewarming is a critical step of the freezing-thawing process.
Molecular control of luteal secretion of progesterone
Authors: Niswender GD
Published: March 2004

Cholesterol provided by low- or high-density lipoprotein is the precursor for biosynthesis of progesterone. Once inside the cell, cholesterol can be used for steroidogenesis or esterified with long-chain fatty acids and stored as cholesterol esters in lipid droplets. When it is needed for steroidogenesis, free cholesterol is transported to the mitochondrion via a mechanism that involves cytoskeletal elements and sterol carrier proteins. Cytochrome P450 cholesterol side chain cleavage enzyme complex converts the cholesterol to pregnenolone, which is then converted to progesterone by 3beta-hydroxysteroid dehydrogenase/delta5,delta4 isomerase in the smooth endoplasmic reticulum. Transport of cholesterol from the cytoplasm to the inner mitochondrial membrane is both the rate-limiting step in progesterone biosynthesis and the step most acutely influenced by second messengers. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptors (PBR) are involved in this transport. StAR may bind cholesterol in the cytosol and transport it to the mitochondrial membrane where PBR is involved in transport from the outer to the inner mitochondrial membrane. Phosphorylation of StAR by protein kinase A (PKA) stimulates cholesterol transport, whereas phosphorylation by PKC may inhibit this process. Endozepine, the natural ligand for PBR, also appears to be involved in regulation of the rate of cholesterol transport to the inner mitochondrial membrane and to play a role in the stimulatory effects of PKA on steroidogenesis. Increased concentrations of endozepine were detected in large luteal cells, and may explain the increased progesterone secretion from this type of cell. Fluorescence energy transfer procedures indicate that StAR associates with PBR in mitochondrial membranes. A model is presented for the proposed interactions of StAR, PBR and endozepine in the transport of cholesterol from the outer to the inner mitochondrial membrane.
Effect of bone morphogenetic protein 2 (BMP2) on oestradiol and inhibin A production by sheep granulosa cells, and localization of BMP receptors in the ovary by immunohistochemistry
Authors: Souza CJ , Campbell BK , McNeilly AS , Baird DT
Published: March 2004

The bone morphogenetic proteins (BMPs) have been implicated in the paracrine regulation of ovarian follicular development. In this study, we investigated the expression of the BMP receptors (BMPRs) in sheep ovaries by immunohistochemistry and the effect of BMP2, a natural ligand for these receptors, on granulosa cells cultured in vitro. Ovaries from cyclic ewes were fixed, embedded in paraffin wax and cut into sections. The sections were rehydrated, submitted to microwave antigen retrieval and treated with polyclonal antibodies against BMPR1A, BMPR1B and BMPR2. Strong immunostaining for all three receptors was observed in the granulosa cell layer of follicles from the primary to late antral stages of development. Staining was also present in the oocyte, corpus luteum, ovarian surface epithelium and, to a lesser extent, the theca layer of antral follicles. For functional studies, granulosa cells were obtained from immature follicles 1-3 mm in diameter. The cells were cultured for 6 days in serum-free medium containing 1 ng oFSH-20 ml(-1) in the presence of 0, 3, 10 or 30 ng ml(-1) human recombinant BMP2. The medium was replaced every 2 days and oestradiol and inhibin A concentrations were measured in the spent medium. In the absence of BMP2, oestradiol and inhibin A production increased as the granulosa cells differentiated in vitro. The addition of the highest dose of BMP2 enhanced oestradiol production (P < 0.05) without affecting the proliferation of the cells. It is concluded that BMP receptors are present in sheep ovaries and that BMPs may have a role in the differentiation of granulosa cells by enhancing the action of FSH.
Expression of vascular endothelial growth factor (VEGF) and its receptors in human endometrium throughout the menstrual cycle and in early pregnancy
Authors: Sugino N , Kashida S , Karube-Harada A , Takiguchi S , Kato H
Published: March 2004

Immunohistochemistry for vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (flt-1) and kinase insert domain-containing region (KDR), was performed on human endometrium obtained from patients with normal menstrual cycles, patients given oestrogen and progesterone, and women in early pregnancy. Intense immunostaining of VEGF was observed in both glandular epithelial and stromal cells during the mid-secretory phase; the immunostaining intensity was increased by administration of oestrogen plus progesterone and strong immunostaining was observed in decidual cells of early pregnancy. In addition to the immunostaining in vascular endothelial cells, strong KDR immunostaining was observed in glandular epithelial cells and in decidualized stromal cells induced by administration of oestrogen plus progesterone, whereas flt-1 immunostaining was negligible. Strong immunostaining for flt-1 and KDR was found in both vascular endothelial cells and decidual cells in early pregnancy. Endometrial stromal cells isolated from proliferative phase endometrium were incubated with oestrogen (10(-8) mol l-1) and medroxyprogesterone acetate (MPA; 10(-6) mol l-1) for 18 days to study the regulation of VEGF, flt-1 and KDR in endometrial stromal cells by oestrogen and progesterone. Expression of VEGF and KDR mRNAs was increased significantly by oestrogen and MPA, accompanied by decidualization, whereas flt-1 mRNA expression was not affected. In conclusion, VEGF and its receptors may play important roles in implantation and maintenance of pregnancy.