The expression and function of the human major histocompatibility complex (MHC) class Ia genes, human leukocyte antigen (HLA)-A, -B, and -C, is well-established; they are expressed in most nucleated cells and present endogenous peptides to CD8 T cells. However, MHC class Ib genes are poorly characterized and have unknown functions. In humans, the best-characterized class Ib gene is HLA-G. This gene has a restricted tissue expression of the mRNA and a unique pattern of protein expression; it is expressed mainly in the extravillous cytotrophoblast cells in the placenta. The function of HLA-G is not clear, but its presence at the maternal-fetal interface suggests a role in protection of the semiallogeneic fetus. Whereas functional studies using in vitro models and transgenic mice provide useful insights regarding the potential function of this molecule, in vivo studies cannot be performed in humans. Nonhuman primates that are closely related to humans phylogenetically contain homologues of HLA-G. The MHC-G loci in nonhuman primates appear to have diverged from the human HLA-G. However, in the rhesus monkey (Macaca mulatta) and olive baboon (Papio anubis), a novel class Ia-related locus has been described. This gene encodes glycoproteins with characteristics that resemble those of HLA-G, including restricted tissue distribution, alternative splicing of mRNA, truncated cytoplasmic domain, and limited polymorphism. Thus, this molecule may be the functional homologue of HLA-G, and these two species may comprise appropriate models for elucidating the function of HLA-G.

The description of healthy individuals homozygous for the human leukocyte antigen-G (HLA-G) null allele raised doubts about the role of HLA-G in fetal-maternal tolerance. In light of recent results, we discuss this point by considering the potential activity of this null allele that might, indeed, produce functional truncated HLA-G molecules. In this context, we have recently described that, like the full-length HLA-G1, the HLA-G2, -G3, and -G4 truncated isoforms may be expressed at the cell surface and may modulate both innate and acquired immune responses.

Within the last few years, there has been a growing interest in the neuroprotective effects of estrogen and the possible beneficial effects of estrogen in neurodegenerative diseases such as stroke, Alzheimer disease, and Parkinson disease. Here, we review the progress in this field, with a particular focus upon estrogen-induced protection from stroke-induced ischemic damage. The important issue of whether clinically relevant selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene and estrogen replacement therapy can exert neuroprotection is also addressed. Although the mechanism of estrogen and SERM neuroprotection is not clearly resolved, we summarize the leading possibilities, including 1) a genomic estrogen receptor-mediated pathway that involves gene transcription, 2) a nongenomic signaling pathway involving activation of cell signalers such as mitogen-activated protein kinases and/or phosphatidylinositol-3-kinase /protein kinase B, and 3) a nonreceptor antioxidant free-radical scavenging pathway that is primarily observed with pharmacological doses of estrogen. The role of other potential mediatory factors such as growth factors and the possibility of an astrocyte role in neuroprotection is also discussed.

Lysophosphatidic acid (LPA) is a prototype of the lysophospholipid mediator family and has multiple effects in the female reproductive system. Although several metabolic routes have been reported for intracellular formation of LPA, a unique route involving lysophospholipase D, an extracellular enzyme that produces LPA in blood and body fluids, is particularly intriguing for its agonistic role. In this study, using an assay with radioactive palmitoyl-lysophosphatidylcholine, we found that lysophospholipase D activity producing palmitoyl-LPA in human serum gradually increased during pregnancy. Elevated activity of lysophospholipase D was not caused by changes in levels of their precursors, lysophosphatidylcholines, in nonpregnant women or in pregnant women at different gestational periods. With increasing length of gestation, the elevated activity in pregnant women was found to produce increasing proportions of LPA with a palmitoyl group versus other LPAs. These results suggest that LPA formed by increased activity of lysophospholipase D in blood might participate in maintenance of pregnancy.

Steroid hormone biosynthesis in the adrenals and gonads is regulated by the steroidogenic acute regulatory (StAR) protein through its action in mediating the intramitochondrial transport of cholesterol. A role for epidermal growth factor (EGF) in modulating steroidogenesis has been previously determined, but the mechanism of its action remains unknown. The present investigation was designed to explore the potential mechanism of action of mouse EGF (mEGF) in the regulation of steroid biosynthesis and StAR protein expression in mLTC-1 mouse Leydig tumor cells. We show that treatment of mLTC-1 cells with mEGF significantly increased the levels of progesterone (P), StAR protein, and StAR mRNA in a time- and dose-dependent manner. The coordinate induction of P synthesis and StAR gene expression by mEGF was effectively inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. Also, longer exposure of mLTC-1 cells to mEGF produced a marked decrease in LH-receptor mRNA expression. These effects of mEGF were exerted through high-affinity binding sites (K_d ∼ 0.53 nmol/L) in these cells. It was also determined that the arachidonic acid (especially lipoxygenase metabolites) and mitogen-activated protein kinase pathways were also involved in the mEGF-induced steroidogenic response. However, involvement of the latter pathway was further assessed in nonsteroidogenic COS-1 cells transfected with the Elk1 trans-reporting plasmids and resulted in a significant increase in luciferase activity in response to mEGF. Furthermore, deletion and mutational analyses demonstrated a predominant involvement of activator protein-1 in addition to the multiple mEGF responsive elements found within the 5′-flanking region (−151/−1 base pairs) of the mouse StAR gene. These findings provide novel insights into the mEGF-induced regulatory cascades associated with steroid synthesis and StAR protein expression in mouse Leydig cells.

Prenatal androgen treatment can alter LH secretion in female offspring, often with adverse effects on ovulatory function. However, female spotted hyenas (Crocuta crocuta), renowned for their highly masculinized genitalia, are naturally exposed to high androgen levels in utero. To determine whether LH secretion in spotted hyenas is affected by prenatal androgens, we treated pregnant hyenas with antiandrogens (flutamide and finasteride). Later, adult offspring of the antiandrogen-treated (AA) mothers underwent a GnRH challenge to identify sex differences in the LH response and to assess the effects of prenatal antiandrogen treatment. We further considered the effects of blocking prenatal androgens on plasma sex steroid concentrations. To account for potential differences in the reproductive state of females, we suppressed endogenous hormone levels with a long-acting GnRH agonist (GnRHa) and then measured plasma androgens after an hCG challenge. Plasma concentrations of LH were sexually dimorphic in spotted hyenas, with females displaying higher levels than males. Prenatal antiandrogen treatment also significantly altered the LH response to GnRH. Plasma estradiol concentration was higher in AA-females, whereas testosterone and androstenedione levels tended to be lower. This trend toward lower androgen levels disappeared after GnRHa suppression and hCG challenge. In males, prenatal antiandrogen treatment had long-lasting effects on circulating androgens: AA-males had lower T levels than control males. The sex differences and effects of prenatal antiandrogens on LH secretion suggest that the anterior pituitary gland of the female spotted hyena is partially masculinized by the high androgen levels that normally occur during development, without adverse effects on ovulatory function.

Uterine decidualization is accompanied by the remodeling of the cell-matrix and cell-cell interactions around the endometrial stromal cells to allow an appropriate invasion of trophoblasts. This remodeling is thought to require the proteolysis of extracellular matrix proteins or cell adhesion molecules; however, the molecular mechanism remains poorly understood. In this study, decidualization induced the expression and activation of an extracellular serine protease neuropsin in the mouse uterus. Although nonpregnant uteri contained little neuropsin, the protein content and enzymatic activity increased markedly and peaked at the midgestational period in pregnant uteri. Neuropsin expression and activity was also upregulated in artificially induced deciduomata but not in nondecidualized pseudopregnant uteri. Neuropsin is the first extracellular protease to show the evident induction of expression and activity by decidualization and might contribute to the remodeling of extracellular components after decidualization.

Intracellular pH recovery in Quackenbush Swiss mouse preimplantation embryos following acid loading was investigated under conditions of H -monocarboxylate cotransporter inactivity. Isoform-sensitive inhibitors of Na -H exchange (NHE) were used to block the Na -dependent component of the response. A biphasic dose-response curve for HOE-694 and N-methylisopropylamiloride (MIA) suggested that two isoforms (putatively NHE1 and NHE3) are active in the oocyte, 1-cell, and 2-cell stages. By the blastocyst stage, loss of one of the MIA-sensitive NHE activities (putatively NHE3) was observed in isolated inner cell masses, and an MIA-resistant component of the recovery was identified. The MIA-resistant component was inhibited by 2 mM amiloride and enhanced by external K and by 4,4′-diisothiocyanostilbene-2,2′-disulfonate, suggesting NHE4 activity. However, unlike NHE4 in other tissues, the MIA-resistant component did not transport Li in exchange for H , and reverse transcription-polymerase chain reaction detected NHE4 mRNA in the oocyte but not in later stages. Trophoblast, whether in intact or collapsed blastocysts, did not show measurable NHE activity or MIA-sensitive activity during recovery from acid load. Both trophoblast and pluriblast manifested an H conductance in response to acid load. This H conductance was first detected at the 8-cell stage and was blocked by zinc in the isolated inner cell mass but not in trophoblast. No other effective inhibitors of its activity were found.

Calcitonin gene-related peptide (CGRP) levels in plasma and the dorsal root ganglia (DRG) are increased during pregnancy and in ovariectomized rats injected with ovarian hormones. Vasodilatory responses to CGRP are also increased in these animals. In the present study, we hypothesized that pregnancy and ovarian hormones elevate the contents of CGRP in perivascular nerves. We assessed CGRP-dependent mesenteric vascular relaxation induced by electrical field stimulation (EFS) and arterial content of CGRP. Because the mesenteric artery represents resistance vessels, segments of mesenteric arteries collected from female rats at different stages of the estrous cycle, pregnancy, or postpartum and from male rats were used in this study. The EFS-induced relaxation in the presence and absence of CGRP_8–37, an antagonist of CGRP, was used to measure CGRP-dependent relaxation, and radioimmunoassay (RIA) of tissue homogenates was used to assess changes in CGRP content in mesenteric branch arteries. The results show that CGRP-dependent, EFS-induced relaxation response was lower in female rats at diestrus and proestrus than in male rats, and no statistically significant differences were observed between Gestational Day 20 and Postpartum Day 2. The RIA revealed significantly lower mesenteric artery CGRP levels in female rats at proestrus, gestation, and postpartum than in female rats at diestrus or in male rats. We conclude that no correlation exists between CGRP-dependent, EFS-induced relaxation and CGRP content in the mesenteric arteries of these animal groups. Because both CGRP levels in DRG and serum are reported to be elevated, the reduced CGRP content in the vasculature during pregnancy and proestrus implicate enhanced basal release of CGRP at the nerve terminal in these animals.

Recently, we found that testicular macrophages produce 25-hydroxycholesterol (25-HC) and express 25-hydroxylase, the enzyme that converts cholesterol to 25-HC. In addition, 25-HC may be an important paracrine factor mediating the known interactions between macrophages and neighboring Leydig cells, because it is efficiently converted to testosterone by Leydig cells. The purpose of the present study was to determine if testosterone can regulate the production of 25-HC in rat testicular macrophages, representing a potential negative-feedback loop from Leydig cells. We found that expression of 25-hydroxylase mRNA and production of 25-HC by cultured testicular macrophages were significantly inhibited by testosterone at 10 μg/ml. This dose of testosterone did not have an effect on cell viability and did not change the rate of mRNA degradation in the presence of actinomycin D. These studies indicate that production of 25-HC is negatively regulated by testosterone, which may be representative of a paracrine negative-feedback loop.

Lactoferrin, an iron-binding glycoprotein, kills bacteria and modulates inflammatory and immune responses. Presence of lactoferrin in the female reproductive tract suggests that the protein may be part of the mucosal immune system and act as the first line of defense against pathogenic organisms. We have discovered that lactoferrin is a major estrogen-inducible protein in the uterus of immature mice and is up-regulated by physiological levels of estrogen during proestrous in mature mice. In the present study, we examined lactoferrin gene expression and its response to estrogen stimulation in the female reproductive tract of several strains of immature mouse, rat, and hamster. The lactoferrin expression in the cycling adult female rat was also evaluated. Lactoferrin gene polymorphism exists among the different mouse strains. In the three inbred mouse strains studied, lactoferrin gene expression is stimulated by estrogen in the immature uterus, although it is less robust than in the outbred CD-1 mouse. We found that the lactoferrin gene is constitutively expressed in the epithelium of the vagina and the isthmus oviduct; however, it is estrogen inducible in the uterus of immature mice and rats. Furthermore, lactoferrin is elevated in the uterine epithelium of the mature rat during the proestrous and estrous stages of the estrous cycle. Estrogen stimulation of lactoferrin gene expression in the reproductive tract of an immature hamster is limited to the vaginal epithelium. The present study demonstrates differential expression and estrogen responsiveness of the lactoferrin gene in different regions of the female rodent reproductive tract and variation among the rodent species studied.

Glucocorticoids are involved in the modulation of the release of parturition hormones from the fetal membranes and placenta, where their actions are determined by the prereceptor glucocorticoid metabolizing enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD). Two distinct isozymes of 11β-HSD have been characterized. In the fetal membranes, 11β-HSD1 is the predominate isozyme; it converts biologically inert 11-ketone glucocorticoid metabolites into active glucocorticoids. Sequence analysis of the cloned 11β-HSD1 gene revealed a putative glucocorticoid response element in the promoter region. However, whether glucocorticoids modulate 11β-HSD1 expression in the fetal membranes is unknown. In this study, 11β-HSD1 and glucocorticoid receptor (GR) were coexpressed in the chorionic trophoblast. Radiometric conversion assay and Northern blot analysis revealed that both 11β-HSD1 reductase activity and mRNA levels were increased by dexamethasone (1 μM, 0.1 μM) in the cultured chorionic trophoblast, and the effects were blocked by GR antagonist RU486 (1 μM). Prior induction of 11β-HSD1 by dexamethasone potentiated the subsequent stimulation of prostaglandin H synthetase 2 expression and secretion of prostaglandin E₂ by cortisone in the chorionic trophoblast. There is colocalization of 11β-HSD1 and GR in the chorionic trophoblast. By binding to GR, glucocorticoids induce the expression of 11β-HSD1 by a possible intracrine mechanism, thereby amplifying the actions of glucocorticoids on prostaglandin production in the fetal membranes. This cascade of events initiated by glucocorticoids may play an important role in the positive feed-forward mechanisms of labor.

Although high amounts of cyclin B1 mRNA are present in bovine oocytes arrested at the germinal vesicle (GV) stage, the protein is not detectable. Furthermore, there is a depletion of the stored cyclin B1 mRNA in the oocyte as follicular growth progresses. To assess the effect of follicular growth on the accumulation of M-phase promoting factor (MPF) components, mRNA and protein levels of cyclin B1 and p34^cdc2 were measured in GV oocytes collected from diverse follicle size groups (<2 mm, 3–5 mm, and >6 mm). Because oocytes collected from very small follicles have high levels of cyclin B1 mRNA, the onset of its accumulation in the oocytes was evaluated by in situ hybridization of fetal ovaries. Also, a comparative expression map of cell cycle-related genes expressed in the oocyte and cumulus cells was established using nylon-based cDNA arrays, which allowed the detection of 35 different genes transcribed mostly in oocytes. Both components of the pre-MPF complex were expressed at the mRNA level in GV oocytes, whereas p34^cdc2 was the only pre-MPF protein detected at that stage, thus indicating that meiosis resumption in bovine oocytes is differentially regulated as compared with other mammals, and meiosis resumption seems to be regulated by the translation of cyclin B1 mRNA.

In mammals, the study of gene expression in the preimplantation embryo has been difficult because the standard procedures used to quantify mRNA generally require large amounts of starting material. The development of protocols using different quantitative strategies generally involving the polymerase chain reaction (PCR) has provided new tools for exploration of gene expression in preimplantation embryos. However, the use of an internal standard, often referred as a housekeeping gene, is essential to normalize the mRNA levels. RNA levels of eight housekeeping genes were quantified using real time PCR throughout the preimplantation period of the bovine embryo to find the most suitable gene to be used as standard. Histone H2a was the best internal standard because the transcript levels were constant across the preimplantation period. Linear amplification of antisense RNA using the T7 promotor for in vitro transcription of the entire RNA pool was evaluated as a suitable way to preamplify the starting material prior to quantification and was effective in providing accurate RNA abundance profiles throughout the preimplantation period. However, the amplification appears to be template dependent because the amplification factors were higher for some genes.

Placental transfer of maternal calcium (Ca² ) is carried out in vivo by the syncytiotrophoblast layer. Although this process is crucial for fetal development, it remains poorly understood. Cytotrophoblasts isolated from human term placenta undergo spontaneous syncytiotrophoblast-like morphological and biochemical differentiation in vitro and are thought to reflect in vivo syncytiotrophoblast. In the present study, we characterized the Ca² uptake potential and the expression of several Ca² channels by human trophoblasts during differentiation in vitro for up to 6 days. Secretion of hCG (specific differentiation marker) and uptake of Ca² by trophoblasts increased gradually as a function of days in culture. Both hCG secretion and Ca² uptake were maximal on Day 4 and declined on Days 5–6. Expression of the Ca² transporter proteins CaT1 and CaT2 was revealed by reverse transcription-polymerase chain reaction in cytotrophoblasts freshly isolated from human term placenta. In addition, messengers for two L-type Ca² channel isoforms (α_1C and α_1D) were also detected. Levels of CaT1, CaT2, and L-type Ca² channel mRNA increased gradually during culture, reaching a maximum between Days 2 and 3. In contrast to CaT1 and CaT2 expression that declined thereafter to levels observed on Day 1, L-type channel expression decreased by 50% but remained above the expression level of Day 1. Our results indicate that the pattern of CaT1 and CaT2 expression correlates with the Ca² uptake potential along the differentiation of cultured human trophoblasts isolated from term placenta. This correlation provides circumstantial evidence for a role of this family of channels in basal Ca² uptake by the syncytiotrophoblast.

The receptors for neurokinin 1 (NK1-R), neurokinin 2 (NK2-R), and neurokinin 3 (NK3-R) are expressed and functionally active in the uterus, promoting strong contractions of the myometrium. Previously, we demonstrated that myometrial contractility activated by the NK-Rs is regulated by estrogen. In the current study, we furthered our investigations of the role of estrogen in the regulation of NK3-R-mediated myometrial contractility. Estrogen promotes both heterologous and homologous desensitization of NK3-R-mediated uterine contractility. In tissue obtained from estrogen-dominated rats (ovariectomized estrogen-treated rats and rats in estrus), the magnitude of uterine contractions decreased in response to consecutive additions of the NK3-R-selective agonist senktide. By addition of the fourth dose of agonist, the contractile response was routinely barely above baseline. In contrast, in tissue obtained from non-estrogen-dominated rats consecutive doses of senktide resulted in contractions of identical magnitude. The homologous desensitization was specific to the NK3-R, and the desensitization of the NK3-R-mediated response did not affect the magnitude or nature of uterine contractions in response to NK1-R or NK2-R activation. Furthermore, heterologous and homologous desensitization of NK3-R-mediated contractility is dependent upon the duration of exposure to estrogen. This complex mechanism appears to be important in intact tissue; capsaicin-mediated release of endogenous neuropeptides resulted in a desensitization of response to subsequent stimulation with senktide in estrogen-dominated uterine tissue.

The genetic manipulation of donor cells before nuclear transfer (NT) enables prior selection for transgene integration. However, selection for genetically modified cells using antibiotic drugs often results in mixed populations, resulting in a mixture of transgenic and nontransgenic donor cells for NT. In this study, we attempted to develop efficient strategies for the generation of human bile salt-stimulated lipase (BSSL) transgenic cows. Preimplantation screening by either biopsy or green fluorescent protein (GFP) expression was used to detect NT-derived BSSL transgenic embryos to ensure that the calf born would be transgenic. We compared the development rates of NT-derived embryos from G418- and GFP-selected donor cells. There were no significant differences (P < 0.001) in cleavage rate (67.2% vs. 60.0%) and blastocyst formation rate (44.9% vs. 41.2%). We also compared the pregnancy rates of the G418/biopsy and GFP preimplantation screened NT-derived blastocysts. The Day 40 pregnancy rate of the G418/biopsy group (40%) was lower than that of the GFP group (57%), but the calf birth rate of the G418/biopsy group (40%) was higher than that of the GFP group (21%). Healthy BSSL transgenic calves were born after both screening processes. This is the first report of biopsy-screened cloned transgenic animals. The results suggest that both selection methods are useful for detecting transgenic NT embryos without negatively affecting their development into viable transgenic offspring.

Successful cryopreservation requires cells to tolerate volume excursions experienced during permeating cryoprotectant equilibration and during cooling and warming. However, prior studies have demonstrated that mouse spermatozoa are extremely sensitive to osmotically induced volume changes. A series of three experiments were conducted 1) to test the efficacy of two commonly used extender media components, egg yolk (EY) and skim milk (SM), in broadening the osmotic tolerance limits (OTL) of ICR and B6C3F1 murine spermatozoa; 2) to determine if the extender components affected sperm plasma membrane permeability coefficients for water and cryoprotective agent (CPA) characteristics; and 3) to test the effects of permeating and nonpermeating CPA on mouse sperm morphology. In experiment 1, sperm samples were added to 150, 225, 300, 450, or 600 mOsm NaCl, EY, SM, sucrose, or choline chloride at 22°C and then returned to isosmotic conditions. In experiment 2, epididymal sperm were preequilibrated in 1 M glycerol (Gly) or 2 M ethylene glycol (EG) prepared in SM extender, abruptly exposed to isosmotic conditions at 22, 15, or 2°C, and the corresponding volume excursions were measured and analyzed. In experiment 3, the effects of permeating CPA (0.3 M EG or dimethyl sulfoxide) or nonpermeating CPA (12% sucrose or 18% raffinose) on sperm morphology (i.e., principle midpiece folding and putative membrane fusion) were evaluated. Experiment 1 showed that spermatozoa from ICR and B6C3F1 mice have effectively broader OTL when exposed to EY or SM extenders. The results of experiment 2 indicated that, for ICR sperm, the activation energy (E_a) for the hydraulic conductivity (L_p) was unchanged in SM extender. However, for B6C3F1 sperm, there were significant differences in E_a of L_p in the presence of Gly and EG. The result of experiment 3 indicated that permeating CPAs damage sperm membrane integrity, causing a high frequency of head-to-tail or tail-to-tail membrane fusion, whereas this occurrence in the presence of nonpermeating CPA was less than 3%. Finally, the results of experiments 1 and 2 were combined in a mathematical model to predict Gly and EG addition and removal in the presence of SM extender, which would prevent mouse sperm membrane damage. These predictions indicated that, for ICR sperm, both Gly and EG may be added and removed in a single step. However, for B6C3F1 spermatozoa, Gly required a two-step addition while EG only required a single step. For removal from B6C3F1 sperm, Gly required a three-step removal process while EG required a two-step removal.

In regressive corpora lutea, apoptosis of luteal cells, expression of monocyte chemoattractant protein-1 (MCP-1), and accumulation of monocytes/macrophages occur. However, whether these three events are correlated and what cell type expresses MCP-1 have yet to be determined. To clarify these issues, we performed histochemical examinations to determine the localization and the numbers of MCP-1 mRNA-containing cells, apoptotic cells, and monocytes/macrophages in corpora lutea of normally cycling rats. We found that the Mcp-1 gene is expressed in nonapoptotic steroidogenic luteal cells. Corpora lutea that contained MCP-1 mRNA-expressing cells increased in number at estrus together with those containing apoptotic luteal cells. When individual corpora lutea at estrus were analyzed, those with many MCP-1-expressing cells contained few apoptotic cells, and vice versa. These results collectively suggest the following pathway for apoptosis- and MCP-1-dependent regression of the corpus luteum: 1) luteal cells are induced to undergo apoptosis at estrus, and the activation of Mcp-1 gene expression follows in nonapoptotic luteal cells; 2) monocytes/macrophages are chemoattracted by MCP-1 toward corpora lutea containing apoptotic luteal cells; and 3) monocytes/macrophages invade corpora lutea and eliminate apoptotic luteal cells by phagocytosis.

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that is important for expression of genes involved in sexual differentiation, testicular and adrenal development, and hormone synthesis and regulation. To better understand the mechanisms required for SF-1 production, we employed transient transfection analysis and electrophoretic mobility shift assays to characterize the elements and proteins required for transcriptional activity of the SF-1 proximal promoter in testicular Sertoli and Leydig cells and adrenocortical cells. Direct comparison of SF-1-promoter activity in testis and adrenal cell types established that a similar set of regulatory elements (an E box, CCAAT box, and Sp1-binding sites) is required for proximal promoter activity in these cells. Further evaluation of the E box and CCAAT box revealed a novel synergism between the two elements and identified functionally important bases within the elements. Importantly, DNA/protein-binding studies uncovered new proteins interacting with the E box and CCAAT box. Thus, in addition to the previously identified USF and NF-Y proteins, newly described complexes, having migration properties that differed between Sertoli and Leydig cells, were observed bound to the E box and CCAAT box. Transient transfection analysis also identified several Sp1/Sp3-binding elements important for expression of SF-1 in the testis, one of which was previously described for expression in the adrenal gland whereas the other two were newly disclosed elements.

Thrombospondin (TSP)-1 and -2 are extracellular matrix glycoproteins that are both antiangiogenic and important in regulating cellular development, differentiation, and function. To evaluate the expression of TSP in follicular and luteal development, ovarian cycles of Sprague-Dawley rats were synchronized and tissues collected daily at stages corresponding to the early antral, ovulatory, early luteal, and late luteal phases of the ovarian cycle. Immunohistochemistry and Western blot analyses demonstrated that TSP-1 protein and its receptor, CD36, were present in the early antral phase and were localized primarily to the granulosa cells of antral follicles. Both proteins were also present immediately after ovulation and were localized to the developing corpus luteum. Messenger RNA for TSP-1 showed a similar pattern, with expression at the early antral and ovulatory phases. Protein and mRNA expression for TSP-2 was relatively delayed compared to TSP-1, although TSP-2 also was expressed in granulosa cells. Both TSP-1 and -2 were increased in response to LH stimulation in vitro, whereas TSP-2 was suppressed by FSH. The temporal pattern of expression of TSP-1, -2, and CD36, which mirrors the active phases of angiogenesis in this experimental model, is compatible with a role for these proteins in the control of ovarian vascularization.

Treatment of spermatozoa with either the nonionic detergent Triton X-100 (TX) or dithiothreitol (DTT) has been suggested to confer enhanced success on intracytoplasmic sperm injection (ICSI) in mice and humans. Here, we attempted to use both reagents together, to our knowledge for the first time, and found that this caused severe chromosomal breaks in paternal pronuclei. We documented this effect further by treating mouse spermatozoa with several combinations of DTT with and without detergent. Spermatozoa were treated with vigorous pipetting to induce membrane disruption or with TX or the ionic detergent mixed alkyltrimethylammonium bromide (ATAB). Swim-up spermatozoa were used as controls. In each treatment, two samples were tested, with or without the addition of DTT during the treatment procedure. In all samples with DTT, protamine reduction was confirmed by the decondensation assay. Sperm nuclei obtained after different treatments were injected into oocytes for cytogenetic analysis, and paternal and maternal chromosomes of the zygote were visualized and examined. We found that the numbers of normal paternal karyoplates resulting from ICSI with spermatozoa treated with either DTT (87%, 153/176), TX (79%, 112/142), or ATAB (85%, 99/116) alone were similar to swim-up controls (92%, 103/112). However, only 22% (23/103) and 40% (59/149) of examined metaphases were scored as normal in TX DTT or ATAB DTT treatments, respectively. Spermatozoa in which the membranes were disrupted by vigorous pipetting in the presence of DTT had a slightly reduced frequency of normal chromosomes (61%, 64/104), whereas those without DTT were normal (79%, 125/159). However, this difference was not statistically significant. When spermatozoa were treated with TX DTT in the presence of EGTA or a mixture of EGTA and EDTA, the frequency of normal chromosomes was 39% (45/114) and 47% (38/81), respectively, suggesting that endogenous sperm nucleases may play a role in chromosomal damage. Our results indicate that simultaneous treatment of spermatozoa with detergent and DTT induces extensive chromosomal breakage and, therefore, should not be attempted in ICSI.

An increase in the concentration of intracellular free Ca² and in the phosphotyrosine content of specific proteins characterizes human sperm capacitation. Whether tyrosine phosphorylation regulates the intracellular free Ca² concentration through modulation of Ca² -ATPase activity or the phosphotyrosine content is under Ca² regulation was investigated using Ca² -ATPase modulators and tyrosine kinase inhibitors. The presence of the Ca² -ATPase-inhibitor thapsigargin during human sperm capacitation caused an increase in the cytoplasmic free Ca² concentration and was associated with an increase in the phosphotyrosine content of specific sperm proteins. Conversely, a decrease in protein tyrosine phosphorylation was observed when gingerol, a Ca² -ATPase activator, was present during the incubation period. On the other hand, thapsigargin had no effect on the phosphotyrosine content or the cytoplasmic Ca² concentration when spermatozoa were incubated in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX). However, the effect of IBMX on phosphotyrosine-containing proteins appears to be a Ca² -dependent phenomenon, because it was partly inhibited in spermatozoa pretreated with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) even though, by itself, BAPTA-AM caused an increase in sperm protein phosphotyrosine content. Tyrosine kinase inhibitors prevented the increase in the phosphotyrosine content without affecting the cytoplasmic free Ca² concentration. Based on these findings, the present study suggests that Ca² -ATPases are involved in the filling of internal Ca² stores, such as the acrosome, and are inhibited later during capacitation. Their inhibition allows an increase in cytoplasmic free Ca² , which is involved in the subsequent increase in the phosphotyrosine content of specific sperm proteins.

The mammalian sperm tail presents a complex organization in which a number of additional structures, namely outer dense fibers and fibrous sheath, surround the central axoneme and are thought to regulate flagellar motility. We have previously described a novel member of the thioredoxin family of proteins with a spermatid specific expression pattern, spermatid-specific thioredoxin-1 (Sptrx-1). We report here the developmental analysis of Sptrx-1 expression during murine spermiogenesis. Immunocytochemical analysis of Sptrx-1 through the different steps of spermiogenesis in rat seminiferous tubule sections showed that its expression begins at step 9, gets progressively stronger until steps 14–16 (where a peak is reached), and then diminishes in steps 17 and 18 until practically no immunolabeling is detected in step 19 spermatid. During its transient expression in spermiogenesis, Sptrx-1 is most concentrated in the periaxonemal compartment of the tail of the elongating spermatid, except in the very last steps (steps 17–19), when periaxonemal labeling disappears and a residual buildup of Sptrx-1 occurs in the shrinking cytoplasmic lobe. Electron microscopic analysis by immunogold labeling pinpointed the localization of Sptrx-1 to the assembling longitudinal columns of the fibrous sheath, whereas the forming ribs of the fibrous sheath were unlabeled. Immunoblotting of isolated fibrous sheath and tails obtained from epididymal or ejaculated sperm of rat and human confirmed our immunocytochemical observation: Sptrx-1 is no longer a component of the mature fibrous sheath. To our knowledge, this is the first report of a protein that specifically associates to the fibrous sheath during development but does not become a permanent structural component. The expression pattern of Sptrx-1 during rat spermiogenesis suggests that it could be part of a nucleation center for the formation of the longitudinal columns and transverse ribs that bridge the latter.

Retinoic acid is necessary for the maintenance of many lining epithelia of the body, such as the epithelium of the luminal surface of the uterus. Administration of estrogen to prepubertal rats induces in these epithelial cells the ability to synthesize retinoic acid from retinol, coincident with the appearance of cellular retinoic acid-binding protein, type two, which is normally present in these cells only at estrus in the mature, cycling animal. Here, we report the isolation, from a cDNA library prepared from uterine mRNA collected at the estrous stage and from a rat mammary adenocarcinoma cell line, of a cDNA that encodes a novel retinol dehydrogenase. A member of the short-chain alcohol dehydrogenase family, the encoded enzyme was capable of metabolizing retinol to retinal when expressed in cells after transfection of its cDNA. When cotransfected with the cDNA of human aldehyde 6, a known retinaldehyde dehydrogenase, the transfected cells synthesized retinoic acid from retinol. Immunohistochemical analysis revealed that the protein was present in the uterine lining epithelium of the mature animal only at estrus, coincident with the presence of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two. Consequently, this novel short-chain alcohol dehydrogenase is an excellent candidate for the retinol dehydrogenase that catalyzes the first step in retinoic acid biosynthesis that occurs in uterine epithelial cells.

Precontact communication between gametes is established by chemotaxis. Sperm chemotaxis toward factor(s) in follicular fluid (FF) has been demonstrated in humans and mice. In humans, the chemotactic responsiveness is restricted to capacitated spermatozoa. Here, we investigated whether sperm chemotaxis to factor(s) present in FF also occurs in rabbits and, if so, whether only capacitated spermatozoa are chemotactically responsive. Chemotaxis assays were performed by videomicroscopy in a Zigmond chamber. We measured chemotactic responsiveness as a function of FF dilution by means of a novel directionality-based method that considers the ratio between the distances traveled by the spermatozoa both parallel to the chemoattractant gradient and perpendicular to it. A peak of maximal response was observed at 10⁻⁴ dilution of FF, resulting in a typical chemotactic concentration-dependent curve in which 23% of the spermatozoa were chemotactically responsive. In contrast, the percentage of cells exhibiting FF-dependent enhanced speed of swimming increased with the FF concentration, whereas the percentage of cells maintaining linear motility decreased with the FF concentration. The percentages of chemotactically responsive cells were very similar to those of capacitated spermatozoa. Depletion of the latter by stimulation of the acrosome reaction resulted in a total loss of the chemotactic response, whereas the reappearance of capacitated cells resulted in a recovery of chemotactic responsiveness. We conclude that rabbit spermatozoa, like human spermatozoa, are chemotactically responsive to FF factor(s) and acquire this responsiveness as part of the capacitation process.

Herpes simplex virus (HSV)-1 has been discovered in placental tissue from spontaneous miscarriages, but reports of transplacental transmission and fetal infection are extremely rare. Previously, we demonstrated that the villous syncytiotrophoblast, which forms a continuous layer between the maternal and fetal circulation, is resistant to HSV entry. Here, we tested our hypothesis that the villous syncytiotrophoblast prevents transplacental transmission of HSV secondary to decreased expression of HSV entry mediators (HveA, HveB, and HveC). In addition, we investigated the ability of HSV to infect extravillous trophoblast cells, which mediate placental attachment to the uterine wall, and the expression of HSV receptors in these cells. We performed fluorescence-activated cell sorting (FACS) analyses and immunostaining to demonstrate that HveA, HveB, and HveC were not expressed in third-trimester villous trophoblast cells. Consequently, villous explants obtained from third-trimester placentas were resistant to infection by a recombinant HSV-1 vector, HSV-1 KOS, but approximately 20% of mesenchymal cells within the villous core were infected when villous explants were pretreated with trypsin to disrupt the villous trophoblast layer. Conversely, FACS analysis and immunostaining demonstrated that extravillous trophoblast cells expressed HveA, HveB, and HveC, and these cells were efficiently infected by HSV vectors. Infection of extravillous trophoblast cells by HSV-1 was not reduced when the cells were pretreated with an antibody against HveA but was partially reduced when the cells were pretreated with antibodies directed against HveB and HveC. Thus, the decreased expression of herpesvirus entry mediators in villous syncytiotrophoblast prevents placental villous infection, thereby limiting maternal-fetal transmission of HSV.

Activating signal cointegrator 1 (ASC-1) has been recently reported as a coactivator of some nuclear receptors. In the present study, we have analyzed the expression of ASC-1 in the mouse testis and investigated its capacity to modulate the transcriptional activity of androgen receptor (AR). We found that although ASC-1 mRNA was ubiquitously expressed at a low level in mouse tissues, a couple of testis-specific mRNAs were expressed in the adult testis. Cloning of one testis-specific variant revealed that the ubiquitous and testis-specific transcripts of ASC-1 share at least the same open reading frame. The expression of the testis-specific ASC-1 mRNAs was developmentally regulated, and the onset of their expression coincided with the initiation of spermatogenesis. In situ hybridization of mouse testis with ASC-1 antisense probe demonstrated predominant expression of ASC-1 in the interstitial Leydig cells that express AR. Moreover, yeast two-hybrid tests and glutathione S-transferase pull-down assays revealed that ASC-1 associates directly with AR and that the hinge domain of AR and a putative zinc-finger motif of ASC-1 are major determinants for their interaction. Transient transfection assays performed by expressing ASC-1 in combination with AR and an androgen-responsive reporter gene showed that ASC-1 moderately alters the induction of the reporter gene. Taken together, these results suggest that ASC-1 may function as an AR coregulator and have a role in testicular functions.

Recently, we have found that the nitrate/nitrite concentrations in preovulatory follicles significantly decrease after hCG injection and that inducible nitric oxide synthase (iNOS) plays a main role in the decrease of the intrafollicular nitric oxide (NO) concentration. The purpose of the present study was to investigate the role of NO on oocyte meiotic maturation and to consider the physiological means of the decrease in intrafollicular NO concentration. Immature rats received 15 IU of eCG, and ovaries were removed under ether anesthesia 48 h later. Each ovary was bluntly divided into five or six pieces containing from four to seven preovulatory follicles under the microscope and then incubated with hCG, aminoguanidine (AG; an iNOS inhibitor), or S-nitroso-l-acetyl penicillamine (SNAP; an NO donor) for 5 h. After incubation, preovulatory follicles were punctured, and germinal vesicle breakdown (GVBD) was observed. Also, cGMP concentrations in these follicles were measured. Next, denuded oocytes were recovered from preovulatory follicles at 48 h after injection of 15 IU of eCG and incubated with SNAP with or without ferrous hemoglobin. Every 30 min up to 12 h, GVBD was observed. Both AG and hCG promoted GVBD, and SNAP prevented this effect. In addition, AG decreased intrafollicular cGMP levels, and the concomitant addition of SNAP prevented this decrease. Finally, SNAP dose-dependently inhibited GVBD in denuded oocyte, and this effect of SNAP was reversed by the addition of hemoglobin. We conclude that the iNOS-NO-(cGMP) axis may play an important role in oocyte meiotic maturation.

Spatiotemporal expression, endocrine regulation, and activation of epidermal growth factor receptor (EGFR) in the hamster ovary were evaluated by immunofluorescence and in situ hybridization localization. Whereas granulosa cells (GC) of primordial through large preantral (stage 6, 7–8 layers GC) follicles had low immunoreactivity, granulosa cells of antral follicles, theca, and interstitial cells had intense EGFR immunoreactivity. EGFR expression in GC of primordial and small preantral follicles increased progressively from estrous through proestrous, but a significant increase occurred in mural GC of antral follicles following the gonadotropin surge. Interstitial cells around small preantral follicles had strong immunofluorescence, and the intensity increased significantly in fully differentiated thecal cells. Distinct EGFR protein was localized in the nucleus of the oocytes and granulosa cells. FSH significantly stimulated EGFR expression in the GC, especially the mural GC, theca, and interstitial cells in hypophysectomized hamster. Estrogen stimulated EGFR expression in preantral GC as well as in interstitial cells. Progesterone and hCG effect was limited to theca and interstitial cells. EGFR expression correlated well with EGFR activation following endogenous or exogenous gonadotropin exposure. Receptor mRNA expression closely followed the protein expression, with increased mRNA expression in mural GC of antral follicles. These results suggest that low levels of EGF signal as a consequence of low levels of receptors in preantral GC may be critical for cell proliferation, but higher receptor density may evoke increased signal intensity due to activation of other intracellular signal pathways, which activate cellular processes related to granulosa, theca, and interstitial cell differentiation. The spatiotemporal cell type and follicle stage-specific expression of receptor mRNA and protein and EGFR activation is critically regulated by gonadotropins and ovarian steroids, primarily estradiol.

The neuroendocrine reproductive and stress axes are known to be closely linked, but the mechanisms underlying these links remain poorly understood. In the ovine brain, GnRH neurons do not contain type II glucocorticoid (GR), progesterone (PR), or α estrogen (ERα) receptors. We sought to determine whether PR, ERα, and GR coexist within the same hypothalamic neurons. A triple immunocytochemical study, involving antisera raised in three different species, was performed on cryostat sections from ovariectomized ewes treated either with estradiol and progesterone or with progesterone alone. All PR-immunoreactive neurons contained ERα, and about 95% of ERα were PR immunoreactive in the preoptic area and arcuate nucleus. Although the PR with ERα colocalization ratio was not affected by the steroid treatments, immunolabeling for PR was weaker in animals that did not receive estradiol. Numerous PR- and ERα-immunoreactive cells contain GR. PR ERα GR-immunoreactive cells represent 70% of PR, 65% of ERα, and 72% of GR in the preoptic area and 70% of PR, 66% of ERα, and 63% of GR in the arcuate nucleus. These results suggest that estrogen, progesterone, and glucocorticoids may influence the activity of the same neurons to modulate both reproductive and stress axes.

Nutrition may affect the balance between immunity and traits such as reproduction or growth. This study examines the effect of low nutrient availability on immunity indices and lactation in captive Iberian red deer. Twelve hinds and their calves were allotted to a food-restricted (50–60% daily energy requirements) or a control group just after calving. Low calorie intake exerted a greater effect on the immunity of calves than on that of hinds. Whereas no difference was found for hinds, calves of the low intake group showed mean immunoglobulin (Ig) levels higher than those on a standard diet, which suggests that Ig level may indicate the level of fighting against pathogens. Serum indices of body condition in calves showed generally positive correlations with milk nutrient production. In contrast, Ig level within each group showed a pattern inverse to that of the other group for early lactation: in the standard diet group, the greater the milk nutrient produced and calf growth, the lower the Ig level; this relationship was inversed in the low-nutrition group. These results suggest that, on a standard diet, high Ig levels may indicate high levels of pathogen fighting paired to poorer body condition. Inversely, once the first barriers of innate immunity are surpassed, only those calves on the low-nutrition group with greater resources would be able to spend more resources to fight infection. Thus, low calorie intake might boost its slowing effect on growth by increasing the costs of infection fighting.

Rainbow trout sexually mature at the end of Year 3. The form of GnRH that controls gonadotropin release in trout is salmon GnRH (sGnRH). In the tetraploid rainbow trout, two genes encode an identical sGnRH peptide. The sGnRH gene-1 produces one mRNA, whereas sGnRH gene-2 can produce more than one. This study asks whether the transcripts and their protein products are expressed in the brain and gonads and whether the pattern correlates with sexual maturity over the final year leading to first spawning. Brain sGnRH mRNA and protein were continuously present throughout the third year. We show for the first time that the long sGnRH-2 mRNA transcript is expressed in neural tissue and not exclusively in gonadal tissue. Expression of the long sGnRH-2 mRNA in the brain coincides with high levels of sGnRH peptide in the brain during a time of increased gonadal growth. Thus, the long sGnRH-2 mRNA in the brain may act to regulate sGnRH production in a stage-specific rather than a tissue-specific manner. In gonads, local sGnRH is thought to play an autocrine/paracrine role in regulating gonadal maturation and spawning. In the maturing gonads, sGnRH gene-1 and -2 are expressed intermittently. Strikingly, sGnRH peptide was not detected in the gonads at any time during Year 3. These results suggest that either the sGnRH transcripts in the gonads are not translated into protein or, if translated, the protein is rapidly released, resulting in gonadal content below 1 fM per fish.

The human placenta is characterized by extensive trophoblast invasion of the uterus. Indeed, extravillous cytotrophoblast cells invade the decidua and the upper third of uterine spiral arteries in the myometrium. This invasion is reflected in situ by the expression of specific markers. In order to study this invasion process, we have established an in vitro culture model of human extravillous trophoblast isolated from first trimester chorionic villi. The aim of this study was to investigate the effect of a composite matrix, the Matrigel required for the culture of this homogenous population of extravillous trophoblasts (EVCT), on their in vitro differentiation. The effect of Matrigel was studied on different markers characterized by immunocytochemistry and by real-time polymerase chain reaction assay of transcripts. In addition, the expression of 12 different matrix metalloproteases and their inhibitors were investigated. We show that human extravillous cytotrophoblasts acquire an invasive phenotype on Matrigel associated with a specific pattern of protease gene expression. This in vitro model will be of interest to study the cellular mechanisms involved in abnormal trophoblast invasion observed in poor placentation and preeclampsia.

For successful implantation, the embryo must develop to the blastocyst stage and the endometrium must attain a state that is receptive to the implanting blastocyst. In rodents, the timing, duration, and hormonal regulation of this receptive state has been well defined. However, the molecular cascade of events involved in the onset of the receptive phase remains unclear. In the present study, we sought to identify genes involved in the onset of the receptivity using the technique of suppressive subtraction hybridization. Herein we report the isolation, cloning, and characterization of a novel gene, uterine sensitization-associated gene-1 (UASG-1), that is preferentially expressed within the maximally sensitized/receptive rat endometrium. USAG-1 mRNA encodes a putative protein of 206 amino acids that contains a possible N-terminal secretion signal and a C-terminal cystine knotlike motif. Northern blot analysis revealed that induction of USAG-1 mRNA was restricted to the Day 5 pregnant or pseudopregnant uterus. In situ hybridization experiments demonstrated that this induction was restricted to the uterine glandular epithelial cells. Given the remarkably tight restriction of its expression, USAG-1 may be involved in the onset of endometrial receptivity for implantation/sensitization for the decidual cell reaction.

The Xmr (Xlr related, meiosis regulated) gene product is abundantly expressed in primary spermatocytes and is notably associated with nonrecombining segments of sex chromosomes in the XY body. Here we determined whether Xmr was expressed in meiotic oocytes. This was done by reverse transcription-polymerase chain reaction and cDNA sequencing, Western blot analysis, and immunocytochemistry. Unexpectedly, no Xmr message was detected in mouse fetal oocytes. Instead, Xlr (X linked, lymphocyte regulated), a closely related gene expressed in fetal thymus cells at the time of antigen-receptor gene rearrangement, was expressed in oocytes throughout meiotic prophase I. These findings indicate a sex-specific expression of two closely related members of the Xlr gene family during meiotic prophase I. The XLR protein may provide a useful marker for studies on chromatin condensation or DNA recombination in oocytes. In addition, because of the localization of the Xlr sequence family on the mouse X chromosome, the human equivalent of Xlr is a candidate gene for premature ovarian failure.