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Showing papers in "Biology of Reproduction in 1994"


Journal ArticleDOI
TL;DR: Evidence from several species indicates that the initial stages of follicular growth proceed very slowly, while the stages after antrum formation are much more rapid, and dominance appears to be maintained by negative feedback effects of products of the dominant follicle on circulating FSH.
Abstract: Evidence from several species indicates that the initial stages of follicular growth proceed very slowly. In contrast, the stages after antrum formation are much more rapid. Atresia seems to be most prevalent as follicles approach the size at which they could be recruited for potential ovulation. Although most follicles become atretic around that stage, a few are recruited into a cohort or wave of follicles that continue to grow beyond the stage at which atresia normally occurs. Next, a species-specific number of follicles is selected for dominance. In some species (e.g. rats, primates, pigs), dominant follicles develop only during the follicular phase and are thus destined for ovulation. In another group of species (e.g. cattle, sheep, horses), recruitment, selection, and dominance occur at regular intervals, but only the dominant follicle present during the follicular phase ovulates. There is evidence that the mechanism that allows some follicles to be recruited for potential dominance/ovulation is a small elevation in basal FSH that, by chance, occurs around the time the follicle would normally begin atresia. Some recruited follicles are saved from atresia for only a short time. Only the dominant follicle(s) selected from among the recruited follicles grows to ovulatory or near-ovulatory size. What determines which follicle(s) becomes dominant is not known, but dominance appears to be maintained by negative feedback effects of products of the dominant follicle on circulating FSH. Selection and dominance are accompanied by progressive increases in the ability of thecal cells to produce androgen and granulosa cells to aromatize androgen to estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

727 citations


Journal ArticleDOI
TL;DR: Investigation of sheep zygote development of amino acids, ammonium, vitamins, and culture of embryos in groups in Synthetic Oviduct Fluid medium supplemented with BSA found indirect evidence that ruminant embryos utilize amino acids to a greater extent than do rodent embryos.
Abstract: The aim of this study was to develop a serum-free culture system that could support high levels of cleavage and blastocyst formation from sheep zygotes developed in vitro. To this end, we investigated the effects on sheep zygote development of amino acids, ammonium, vitamins, and culture of embryos in groups in Synthetic Oviduct Fluid (SOF) medium supplemented with BSA (32 mg/ml). The inclusion of amino acids in the culture medium had no effect on the percentage of embryos arrested at the 8-16-cell stage when embryos were cultured singly in the same drop of medium for 6 days (43% in SOF; 41% in SOF+amino acids). However, in medium containing all Eagle's amino acids, replacing the culture medium every 48 h to alleviate ammonium toxicity significantly decreased the number of arrested embryos (6%; p < 0.05) and significantly increased blastocyst cell number (52 cells in SOF; 105 cells in SOF+amino acids; p < 0.01) and the number of embryos developing to the blastocyst stage (29% in SOF; 67% in SOF+amino acids; p < 0.05). When the medium was renewed every 48 h, nonessential amino acids and glutamine also significantly decreased the number of arrested embryos (p < 0.05). Culturing embryos singly or in groups in SOF medium with all Eagle's amino acids that was renewed every 48 h resulted in significant increases in blastocyst hatching and mean cell number (47%, 31%, and 79%; 105, 136, and 173 cells for embryos cultured singly, in groups of 2, and in groups of 4, respectively). After culture in groups of 4, blastocyst cell numbers were equivalent to in vivo-developed controls (160 cells) and significantly greater than those developed in serum (103 cells; p < 0.01). Analysis of blastocyst metabolism, expressed on a per-cell basis, revealed that amino acids did not affect either glucose uptake or lactate production, whereas the addition of amino acids and vitamins resulted in a significant increase in both parameters (p < 0.01). A similar response was observed in serum-derived blastocysts. Ammonium production by sheep blastocysts after culture in the presence of amino acids was significantly greater than that produced by mouse blastocysts, indirect evidence that ruminant embryos utilize amino acids to a greater extent than do rodent embryos.(ABSTRACT TRUNCATED AT 400 WORDS)

558 citations


Journal ArticleDOI
TL;DR: This presentation reviews current information on the events that lead to rupture of an ovarian follicle and contains a summary of the morphological changes that occur at the apex of a follicle wall during ovulation.
Abstract: This presentation reviews current information on the events that lead to rupture of an ovarian follicle. It contains a summary of the morphological changes that occur at the apex of a follicle wall during ovulation. Existing information shows that the tenacious connective tissue layers of the tunica albuginea and theca externa must be weakened before the follicle wall can dissociate and break open under the force of a modest intrafollicular pressure. These changes are probably dependent on transformation of quiescent thecal fibroblasts into proliferating cells in a manner that is characteristic of tissue responses to inflammatory reactions. The metabolic factors that initiate transformation of the fibroblasts are uncertain, but they are probably generated by gonadotropin-induced changes in the theca interna ancPgranulosa of a follicle as these layersbegin to luteinize during the ovulatory process.

461 citations


Journal ArticleDOI
TL;DR: The view that depletion of the NGF pool is caused mainly by atresia in younger women but mainly by entrance of NGF into the growing pool in older women is supported.
Abstract: The effect of aging on the number of non-growing follicles (NGF) and early-growing follicles (EGF) was studied in humans through use of a database obtained by pooling two subsets of ovarian pairs (2 x 43 pairs) collected in two distinct populations. A previously suggested model of exponential regression of NGF counts in relation to the subject's age was tested but did not adequately fit the observed data points. This lack of fit is attributable mainly to the existence of a significant relation between a woman's age and the corresponding NGF count decay rate. Consequently, various regression models were tested. Two different periods of decay rate were observed for each population of small follicles. The first corresponds to younger ages with a decay rate that is slow for both types of follicles, although faster for NGF than for EGF. The second period corresponds to older ages with an accelerated decay rate that appears similar for NGF and EGF. The changing points were found at 38.0 +/- 2.4 and 39.0 +/- 1.9 yr (mean +/- SD) for NGF and EGF, respectively. Extrapolation of the fitted model suggested the presence of approximately 402,000 healthy NGF per ovary at birth and a total exhaustion of the follicular stock at around 74 yr of age. These results support the view that depletion of the NGF pool is caused mainly by atresia in younger women but mainly by entrance of NGF into the growing pool in older women. The mechanisms triggering accelerated entrance into the growth phase of NGF are discussed in relation to the previously reported increase in FSH plasma levels that starts in the late thirties, approximately, and precedes the menopausal period by several years.

397 citations


Journal ArticleDOI
TL;DR: Noninvasive fecal assays were used to study steroid metabolism and ovarian activity in several felid species and indicated that steroid metabolism mechanisms appear to be conserved among these physically diverse, taxonomically related species.
Abstract: Noninvasive fecal assays were used to study steroid metabolism and ovarian activity in several felid species. Using the domestic cat (Felis catus) as model, the excretory products of injected [14C]estradiol (E2) and [14C]progesterone (P4) were determined. Within 2 days, 97.0 +/- 0.6% and 96.7 +/- 0.5% of recovered E2 and P4 radioactivity, respectively, was found in feces. E2 was excreted as unconjugated estradiol and estrone (40%) and as a non-enzyme-hydrolyzable conjugate (60%). P4 was excreted primarily as non-enzyme-hydrolyzable, conjugated metabolites (78%) and as unconjugated pregnenolone epimers. A simple method for extracting fecal steroid metabolites optimized extraction efficiencies of the E2 and P4 excretion products (90.1 +/- 0.8% and 87.2 +/- 1.4%, respectively). Analysis of HPLC fractions of extracted fecal samples from the radiolabel-injected domestic cats revealed that E2 immunoreactivity coincided primarily with the unconjugated metabolized [14C]E2 peak, whereas progestogen immunoreactivity coincided with a single conjugated epimer and multiple unconjugated pregnenolone epimers. After HPLC separation, similar immunoreactive E2 and P4 metabolite profiles were observed in the leopard cat (F. bengalensis), cheetah (Acinonyx jubatus), clouded leopard (Neofelis nebulosa), and snow leopard (Panthera uncia). Longitudinal analyses demonstrated that changes in fecal E2 and P4 metabolite concentrations reflected natural or artificially induced ovarian activity. For example, severalfold increases in E2 excretion were associated with overt estrus or exogenous gonadotropin treatment, and elevated fecal P4 metabolite concentrations occurred during pregnant and nonpregnant (pseudopregnant) luteal phases. Although overall concentrations were similar, the duration of elevated fecal P4 metabolites during pseudopregnancy was approximately half that observed during pregnancy. In summary, steroid metabolism mechanisms appear to be conserved among these physically diverse, taxonomically related species. Results indicate that this hormone-monitoring approach will be extremely useful for elucidating the hormonal regulatory mechanism associated with the reproductive cycle, pregnancy, and parturition of intractable and endangered felid species.

373 citations


Journal ArticleDOI
TL;DR: The total cell number of KSOM-cultured blastocysts indicates that 5-6 cell divisions are possible when embryos are cultured for 96 h from pronuclear zygotes, a significant improvement in performance over that of other defined media for the culture of zygote to blastocyst stage.
Abstract: We have recently reported the use of sequential simplex optimization methods to design a medium (SOM) that overcomes the block to development beyond two cells which occurs in vitro in embryos from an outbred strain of mouse. We have examined this medium and several others for their ability to foster development of CF1 female x B6D2F1 male mouse embryos through the blastocyst stage. A modification of medium SOM, designated KSOM, with an increased concentration of K+ (2.5 mM), also supports growth beyond the two-cell block; compared to other media tested it produces a higher rate of compaction (100%), provides a larger yield of blastocysts (88%), and stimulates an increased rate of cell division of the trophoblast cells. The total cell number of KSOM-cultured blastocysts (44 +/- 12; n = 30) indicates that 5-6 cell divisions are possible when embryos are cultured for 96 h from pronuclear zygotes. This is a significant improvement in performance over that of other defined media for the culture of zygotes to blastocysts.

360 citations


Journal ArticleDOI
TL;DR: To assess the stage at which mosaicism occurred in preimplantation human embryos through use of fluorescence in situ hybridization (FISH) with multiple probes, full karyotypes of blastomeres from 2- to 8-cell human embryos are obtained.
Abstract: In the human, mosaicism may occur before implantation; but, to determine when it first occurs, it is necessary to study the chromosomal complement of all blastomeres. Full karyotypes of blastomeres from 2- to 8-cell human embryos by conventional karyotyping of metaphase spreads are difficult to obtain. The aim of this study was to assess the stage at which mosaicism occurred in preimplantation human embryos through use of fluorescence in situ hybridization (FISH) with multiple probes. All or most blastomeres from 2- to 12-cell human embryos were analyzed by FISH using probes for gonosomes and chromosome 18. FISH was performed on blastomeres from 117 morphologically normal monospermic embryos that were not transferred after preimplantation diagnosis because of their risk of carrying X-linked disease; 20 (17.1%) of these embryos were mosaic. Another group of 163 arrested or morphologically abnormal monospermic embryos were also analyzed by FISH; 47 (28.8%) of these embryos were mosaic. In addition, 37 dispermic embryos were analyzed, and 28 (75.7%) of these were found to be mosaic. Mosaicism first occurred at the second cleavage division when the monospermic embryo was mostly diploid and at the first cleavage division when the embryo was mostly haploid, polyploid, or dispermic.

278 citations


Journal ArticleDOI
TL;DR: Spindles did not return to normal in most oocytes regardless of cooling temperature or rewarming scheme, and step-wise rewarming was no more beneficial than direct rewarming.
Abstract: The purpose of this study was to evaluate effects of cooling and rewarming on the meiotic spindle apparatus of bovine oocytes In experiment 1, in vitro-matured bovine oocytes were either maintained at 39 degrees C or cooled abruptly to 4 degrees C or approximately 25 degrees C Immunohistochemical and DNA staining for visualization of microtubules and chromosomes, respectively, revealed an anastral, barrel-shaped spindle in bovine oocytes Exposure to 4 degrees C for 10-20 min caused complete disappearance of the spindle Some chromosome dispersion occurred after 60 min at 4 degrees C After exposure to approximately 25 degrees C for 30 min, 90% of oocytes appeared abnormal, having either an abnormal spindle or no spindle In experiment 2, oocytes cooled to either approximately 25 degrees C or 4 degrees C for 30 min were rewarmed directly or in steps for 15 or 60 min Spindles did not return to normal in most oocytes regardless of cooling temperature or rewarming scheme Step-wise rewarming was no more beneficial than direct rewarming More of the oocytes rewarmed directly contained dispersed chromosomes as time at 39 degrees C increased

268 citations


Journal ArticleDOI
TL;DR: This study is the first demonstration of the presence of flt on non-endothelial cells in vivo and suggests a role for VEGF in the growth and differentiation of cytotrophoblast at implantation.
Abstract: Vascular endothelial growth factor (VEGF; also known as vascular permeability factor) is a secreted angiogenic growth factor. It is highly specific for endothelial cells, and its receptor, the fms-like tyrosine kinase (flt), has been localized only to endothelial cells in vivo. Here we describe the expression of mRNA encoding flt in human trophoblast as revealed by in situ hybridization. This mRNA is highly expressed in the cytotrophoblast shell and columns and also highly expressed by the extravillous trophoblast (EVT) in the maternal decidua both in the first trimester and at term. The trophoblast-like choriocarcinoma cell line BeWo also expresses this receptor and the related receptor, kinase domain-containing receptor (KDR), which is also a receptor for VEGF. Treatment of the cell line BeWo with VEGF165 stimulated 3H-thymidine incorporation and tyrosine phosphorylation of MAP (mitogen-activated protein) kinase in a time- and dose-dependent fashion. This study is the first demonstration of the presence of flt on non-endothelial cells in vivo and suggests a role for VEGF in the growth and differentiation of cytotrophoblast at implantation.

250 citations


Journal ArticleDOI
TL;DR: The results indicate that immature mouse oocytes possess intracellular stores of releasable Ca2+ similar in size toCa2+ stores in eggs; however, these stores are less sensitive to IP3.
Abstract: Fertilization of the immature, prophase I-arrested mouse oocyte produces multiple Ca2+ transients similar to those of the mature, metaphase II egg; however, the first Ca2+ transient is much lower in amplitude and shorter in duration. In contrast to prophase I-arrested oocytes, maturing oocytes fertilized after germinal vesicle breakdown have first Ca2+ transients similar to those of mature fertilized eggs. Immature, prophase-arrested oocytes release less Ca2+ in response to injection of inositol 1,4,5-trisphosphate (IP3) than eggs. At high concentrations, the sulfhydryl reagent, thimerosal (200 microM), causes Ca2+ oscillations in eggs and produces similar oscillations in oocytes. A lower concentration of thimerosal (25 microM) does not cause Ca2+ oscillations, but does sensitize IP3-induced Ca2+ release in both eggs and oocytes, since IP3-induced Ca2+ release is enhanced in the presence of 25 microM thimerosal. Incubation of oocytes in 25 microM thimerosal before injection of 2.2 microM IP3 causes oocytes to release as much Ca2+ as is released in eggs injected with 2.2 microM IP3. These results indicate that immature mouse oocytes possess intracellular stores of releasable Ca2+ similar in size to Ca2+ stores in eggs; however, these stores are less sensitive to IP3. Development of the IP3-induced Ca2+ release mechanism may be an important component of maturation; at fertilization of the egg, Ca2+ must be elevated to levels sufficient to activate further development and establish a block to polyspermy. Mouse oocytes appear to develop an increased sensitivity to IP3 during the course of oocyte maturation.

222 citations


Journal ArticleDOI
TL;DR: In situ analysis of DNA fragmentation in testes of unilaterally cryptorchid rats at 7 days after the operation indicated that germ cells, mainly primary spermatocytes were affected and that the percentage of seminiferous tubules containing labeled cells increased in the operated testis as compared to the contralateral control in the same animal.
Abstract: Although surgical induction of cryptorchidism in the rat is known to cause infertility due to disruption of spermatogenesis, the exact cellular mechanism responsible for the degenerative changes in cryptorchid testes is unclear. Using a sensitive autoradiographic method for the detection of apoptotic DNA fragmentation, we have investigated the effect of experimentally induced cryptorchidism on apoptotic cell death in testes of immature rats. Bilateral or unilateral cryptorchidism decreased the weight of affected testes within 4 days; these decreases (24-27%) became significant (p 15 kb), whereas DNA cleavage into low molecular weight ladders characteristic of apoptosis was increased by induction of bilateral cryptorchidism in a time-dependent manner, i.e., 2.0-, 2.8-, and 4.2-fold (p < 0.05) at 2, 4, and 7 days after operation, respectively. In unilaterally cryptorchid animals, sham-operated testes also contained predominantly high molecular weight DNA, whereas induction of cryptorchidism of the contralateral testes increased DNA cleavage into low molecular weight fragments 3.0-, 2.8-, and 3.9-fold (p < 0.05) at 2, 4, and 7 days after the operation, respectively. In situ analysis of DNA fragmentation in testes of unilaterally cryptorchid rats at 7 days after the operation indicated that germ cells, mainly primary spermatocytes, were affected and that the percentage of seminiferous tubules containing labeled cells increased in the operated testis as compared to the contralateral control in the same animal. Furthermore, according to radioligand receptor analysis, specific ['251]-hCG binding to cryptorchid testes was not affected at 2 and 4 days after the operation but was significantly (p < 0.05) decreased (32-35%), in relation to values in sham-operated testes in both bilaterally and unilaterally cryptorchid animals, at 7 days after surgery. In addition, testis ['25I]-hCG binding per milligram protein was not affected by the induction of bilateral or unilateral cryptorchidism throughout the 7 days of the experiment. Our data indicate that cryptorchidism-induced testis cell degeneration is mediated by apoptosis probably as the result of increases in testis temperature, leading to delayed decreases in LH receptors of Leydig cells. Although apoptotic cell death induced by bilateral cryptorchidism might be affected by changes in systemic factors, the increase of apoptosis in male germ cells after unilateral cryptorchidism is presumably regulated by local testicular factors. Experimentally induced cryptorchidism provides a useful model for study of the role of elevated temperature on testis cell apoptosis.

Journal ArticleDOI
TL;DR: The data demonstrate that the sensitivity of porcine embryos to chilling is related to their high lipid content and that they can become tolerant to chilling if their lipid content is reduced.
Abstract: The lipid content of porcine 1-cell stage embryos was reduced (delipated) through the use of micromanipulation to remove the lipid layer formed after centrifugation Of 94 delipated embryos chilled to 4 degrees C for 1 h at the 1-cell or 2- to 4-cell stage, 60 (64%) cleaved in culture with development to the morula-blastocyst stage, whereas all of the control embryos lysed within 24 h Significantly more embryos developed beyond the 8-cell stage when they were chilled at the 2- to 4-cell stage compared with chilling at the 1-cell stage (44%, 20 of 45 vs 18%, 9 of 49) Fewer embryos developed after chilling if they were only partially rather than fully delipated Developmental rates of partially delipated embryos to the 8-cell and blastocyst stages were 33% (13 of 40) and 8% (3 of 40), rates significantly (p or = blastocyst: 40%, 19 of 48 vs 57%, 17 of 30) These data demonstrate that the sensitivity of porcine embryos to chilling is related to their high lipid content and that they can become tolerant to chilling if their lipid content is reduced

Journal ArticleDOI
TL;DR: The role of the sympathetic and parasympathetic branches of the autonomic nervous system control of different aspects of rat prostate growth and atrophy is examined.
Abstract: Many factors are implicated in the development of prostatic growth: androgens, growth factors, and stromo-epithelial interaction. This study examines the role of the sympathetic and parasympathetic branches of the autonomic nervous system control of different aspects of rat prostate growth and atrophy. Unilateral sympathectomy leads to decreases in ventral prostate weight, DNA, and protein content in the lesioned side. Unilateral parasympathectomy leads to increases in ventral prostate weight, DNA, and protein content in the intact side. The separate effects of sympathectomy and parasympathectomy are maintained across a diverse combination of neural manipulations. Significant re-innervation does not occur by 60 days after manipulation as assessed by tissue norepinephrine levels. There appears to be a differential effect of the autonomic nervous system on growth and maintenance of the ventral prostate. The mechanism of contralateral hyperplasia and ipsilateral atrophy has potential significance in understanding human abnormal prostate growth.

Journal ArticleDOI
TL;DR: The results show that the expression and functional properties of HSF2 are regulated in spermatogenic cell types of the mouse testis, supporting a role for this transcription factor as a regulator of hsp gene expression during sperMatogenesis.
Abstract: We have examined the expression and function of heat shock transcription factor 2 (HSF2) in spermatogenic cells of mouse testis The results of in situ RNA hybridization analysis, RNA filter hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicate that HSF2 mRNA expression in testis is subject to developmental and cell type-dependent, as well as stage-dependent, regulation Localized expression of HSF2 mRNA in testis first appears between Day 14 and Day 21 of postnatal development In adult testis, HSF2 mRNA is found at highest levels in spermatocytes and round spermatids Immunocytochemical staining and gel mobility shift analysis demonstrate that HSF2 protein is localized to the nuclei of spermatocytes and round spermatids and that this transcription factor exists in testis in a constitutively active DNA-binding state We further demonstrate that the constitutive HSF2 DNA-binding activity present in testis is able to interact with promoter sequences of the hsp702 gene, a testis-specific member of the hsp70 gene family Taken together, our results show that the expression and functional properties of HSF2 are regulated in spermatogenic cell types of the mouse testis, supporting a role for this transcription factor as a regulator of hsp gene expression during spermatogenesis

Journal ArticleDOI
TL;DR: The mechanisms involved in maternal recognition of pregnancy are very diverse between species and may involve direct luteotropic stimulation of the CL, reduced uterine secretion of P GF2 alpha, and/or inhibition of actions of PGF2 alpha at the level of theCL.
Abstract: A number of morphological and biochemical changes occur as the cells of the recently ovulated follicle luteinize and develop into a functional CL. There are two distinct steroidogenic luteal cell types that appear to differentiate from thecal and granulosal cells in the follicle. The control of progesterone secretion is quite different in the two cell types. Prostaglandin F2 alpha (PGF2 alpha) is the primary luteolytic hormone in most mammals. PGF2 alpha appears to exert its antisteroidogenic actions via activation of the protein kinase C system, while its cytotoxic effects appear to be mediated via a dramatic increase in intracellular levels of free calcium. The mechanisms involved in maternal recognition of pregnancy are very diverse between species and may involve direct luteotropic stimulation of the CL, reduced uterine secretion of PGF2 alpha, and/or inhibition of actions of PGF2 alpha at the level of the CL.

Journal ArticleDOI
TL;DR: This study represents a systematic analysis of apoptosis in the rat ovary at different functional stages and supports the hypothesis that apoptosis is involved in the process of follicular atresia.
Abstract: Apoptosis is a type of physiologic cell death that occurs in many tissues and be regulated by peptide growth factors. Recent studies indicate that apoptosis occurs in the ovary during follicular atresia in several animal species, including the rat, pig, chicken, baboon, and rabbit. The purpose of this study was to demonstrate, through in situ identification of apoptotic cells in intact ovarian sections, the sites in which apoptosis occurs in the rat ovary in different functional states. We evaluated the presence of apoptosis in three models: immature rats, eCG-treated rats and adult cycling rats. Paraffin ovarian sections were pretreated with proteinase K and then end-labeled with biotinylated deoxyuridine triphosphate (dUTP) by incubation with the enzyme terminal deoxynucleotidyl transferase (TDT). They were then stained through use of avidin-conjugated peroxidase with 3,3'-diaminobenzidine as the substrate. Healthy antral and preantral follicles had no staining. The nuclei of granulosa cells of preantral and antral atretic follicles were positively stained in all the animal groups. Scattered theca cells were also stained. Stromal cells were consistently negative. Positive controls were sections pretreated with DNase I; these displayed intense staining of all nuclei. Negative controls, in which either terminal TDT or its biotinylated substrate was omitted, were appropriately negative. This study represents a systematic analysis of apoptosis in the rat ovary at different functional stages and supports the hypothesis that apoptosis is involved in the process of follicular atresia.

Journal ArticleDOI
TL;DR: Findings suggest that, in humans, LIF plays a role in uterine function during the menstrual cycle, as well as during pregnancy.
Abstract: Leukemia inhibitory factor (LIF), a cytokine that induces macrophage differentiation in the murine Ml myeloid leukemia cellline, is essential for blastocyst implantation in mice However, its expression and the role it plays in the human uterus areunknown To clarify these issues, we examined LIF gene expression in the human uterus by Northern blot hybridization and bya quantitative reverse transcription-polymerase chain reaction (RT-PCR) method Analysis of LIF mRNA showed two hybridizationbands, with estimated mRNA sizes of about 40-kb pairs and 18-kb pairs LIF mRNA was detected at high levels in endometrialtissue and decidua, but at low levels in the chorionic villus in first trimester and term placenta In the secretory phase, theendometrial tissue showed higher LIF expression than in the proliferative phase (95-fold; p < 001) The endometrial tissueswere separated into a stroma-enriched fraction (SF) and an epithelium-enriched fraction (EF), and the LIF mRNA levels in eachfraction were examined by quantitative RT-PCR These levels were higher in the EF than in the SF (33-fold; p < 005) Thesefindings suggest that, in humans, LIF plays a role in uterine function during the menstrual cycle, as well as during pregnancy

Journal ArticleDOI
TL;DR: The data indicate that preimplantation embryos are very sensitive to conditions that can cause oxidative stress and show also that their glutathione status changes dramatically during development.
Abstract: Experiments were conducted to elucidate the status of glutathione present in the oxidized (GSSG), reduced (GSH), and protein-mixed disulfide (GSSprotein) forms in preimplantation mouse embryos during development and after treatment with tertiary-butyl hydroperoxide (tBH) to cause oxidative stress. Glutathione was measured at picomolar levels by fluorimetric HPLC after derivatization of extracted embryonic samples with dansyl chloride. GSH content decreased approximately 10-fold from that in the unfertilized oocyte to 0.12 pmol/blastocyst, representing an estimated change in concentration from 7 to 0.7 mM. GSH levels were lower in embryos cultured in vitro than in embryos that developed in vivo. Addition of GSH to the culture medium improved in vitro development of mouse embryos, but surprisingly the addition of glutathione monoethyl ester did not. Addition of low levels of the oxidant tBH (13.2 microM) to culture medium decreased the percentage of two-cell and blastocyst stage embryos that exhibited further development. After 15-min exposure to 13.2 microM tBH, GSH levels were markedly decreased in the two-cell stage embryo (75%), but only slightly decreased (25%) in the blastocyst. The loss of GSH was accounted for by increases in GSSG and GSSprotein, indicating that the embryo was undergoing oxidative stress. These data indicate that preimplantation embryos are very sensitive to conditions that can cause oxidative stress and show also that their glutathione status changes dramatically during development.

Journal ArticleDOI
TL;DR: RecZP3 induced the sperm acrosome reaction and promoted fusion of human spermatozoa with zona-free hamster oocytes, indicating that the recombinant protein is biologically active, and provides an attractive tool for studying the initial stage of the human fertilization process.
Abstract: Studies on the role of specific molecules in the human fertilization process and additional assessments of potential applications for these proteins are hampered by the limited amount of available biological material. However, this drawback might be circumvented by the recent cloning of several gamete-specific genes, which opens possibilities for the production of recombinant proteins. By use of cDNA and genomic DNA fragments of the human ZP3 gene, which encodes a major constituent of the zona pellucida surrounding the oocyte, a 2.7-kb minigene was constructed containing the natural third and fourth introns of the gene and a truncated intron between exons 2 and 3. This ZP3 DNA was transfected to Chinese hamster ovary cells, and a single-cell clone producing the recombinant ZP3 protein (recZP3) was generated. Western blot analysis of culture medium from these cells showed that recZP3 has a molecular mass + 5 kDa smaller than that of natural ZP3. Under reducing conditions, it migrates at an apparent molecular mass of 55-60 kDa. RecZP3 induced the sperm acrosome reaction and promoted fusion of human spermatozoa with zona-free hamster oocytes, indicating that the recombinant protein is biologically active. RecZP3 provides an attractive tool for studying the initial stage of the human fertilization process. Furthermore, it might have clinical applications in the development of diagnostic tests for male infertility and serve as target antigen in the design of contraceptive vaccines.

Journal ArticleDOI
TL;DR: This is the first time that sperm chemotaxis and chemokinesis in response to a follicular factor(s) in mammals has been established and has been distinguished from other processes that might cause sperm accumulation.
Abstract: Human spermatozoa accumulate in vitro in diluted follicular fluids obtained from follicles from which the eggs have been fertilized. Using capillary assays under a variety of experimental conditions (ascending or descending gradients of follicular fluid, or no gradient at all) and microscopic assays in which individual spermatozoa could be followed, we found that the sperm accumulation in follicular fluid was the result of both sperm chemotaxis and chemokinesis and eventually hyperactivation-like motility. We determined the optimal conditions for sperm accumulation, which involved sperm preincubation (possibly to induce sperm capacitation) and proper dilution of follicular fluid. In all the assays, the net accumulation was low, probably reflecting the chemotactic responsiveness of only a small fraction of the sperm population at any given time. We partially fractionated follicular fluid in a Centricon microconcentrator (Amicon, Danvers, MA) and by acetone precipitation, and found that at least one of the chemotactic factors is a small (< 10-kDa) molecule that is probably nonhydrophobic. This is the first time that sperm chemotaxis and chemokinesis in response to a follicular factor(s) in mammals has been established and has been distinguished from other processes that might cause sperm accumulation. The physiological significance of these findings is discussed.

Journal ArticleDOI
TL;DR: Luteal growth from stage I to stage II resulted from cell proliferation as shown by a high LI at stage I, accompanied by increased luteal DNA content but no change in average cell size, and by similar protein: DNA ratios.
Abstract: To evaluate the kinetics of luteal growth, bovine CL were obtained from four stages (stage I, Days 1-4; stage II, Days 5-10; stage III, Days 11-17; stage IV, Days 18-21) of the estrous cycle, and luteal fresh weight as well as DNA, protein, and progesterone contents was determined. To evaluate the relative rate of cell proliferation, proliferating cell nuclear antigen (PCNA; a specific marker for cell proliferation) was immunolocalized in paraffin-embedded luteal tissue sections. To evaluate the relative rate of cell death, nucleosomal fragmentation of DNA (a specific marker for apoptosis) was detected by agarose gel electrophoresis and also by histochemical localization in paraffin-embedded luteal tissue sections. Luteal fresh weight and DNA, protein, and progesterone contents increased (p < 0.01) from stage I to stage II, were similar between stages II and III, and then decreased (p < 0.01) from stage III to stage IV. The ratio of protein to DNA (an index of average cell size) was similar for stages I, II, and III and then decreased (p < 0.01) at stage IV. For stage I (corpora hemorrhagica), most proliferating (PCNA-positive) cells were located in or around the core of the tissue infoldings (presumably thecal-derived areas), whereas relatively few proliferating cells were located at the periphery of the tissue infoldings (presumably granulosa-derived areas). For stages II, III, and IV, the majority of proliferating cells appeared to be small cells (i.e., small parenchymal cells, fibroblasts, and endothelial cells). The labeling index (LI; percentage of cells that were PCNA-positive) was greatest at stage I (20.3 +/- 1.1%); it then decreased (p < 0.01) by stage II and was similar at stages II, III, and IV (3.4 +/- 1.1%). Apoptosis, as determined by evaluation of nucleosomal DNA fragmentation by agarose gel electrophoresis and in situ localization, was detectable only at stage IV. These data demonstrate that luteal growth from stage I to stage II resulted from cell proliferation as shown by a high LI at stage I, accompanied by increased luteal DNA content but no change in average cell size, and by similar protein: DNA ratios. Luteal regression from stage III to stage IV was primarily associated with cell deletion and decreased cell size as shown by a decrease in luteal DNA content and the appearance of apoptosis along with a decrease in the luteal protein: DNA ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

Journal ArticleDOI
TL;DR: The presence of significantly greater amounts of the mRNA during the late follicular phase of the menstrual cycle is consistent with the proposed estrogen control and isolated the complete cDNA for a human oviductal glycoprotein.
Abstract: A 120-kDa oviduct-specific glycoprotein is synthesized and secreted into the oviductal lumen during estrogen dominance in the human. The objective of this investigation was to clone, sequence, and characterize the cDNA to this core protein. Rapid amplification of cDNA ends was used to clone a contiguous 3' cDNA end and 5' cDNA end. The total length of the cDNA was determined to be 2.2 kb by sequence analysis and exhibited a 92% sequence identity with the comparable overlapping baboon cDNA (1.2 kb). A high degree of homology was found to the N-terminal sequence of hamster oviductin and the partial sequence of a homologous baboon and bovine oviduct glycoprotein. Northern blots revealed a single mRNA species of 2.4 kb. Using RNA from various tissues of an estrogen-treated baboon, we found that the mRNA for the oviductal glycoprotein was present only in the oviduct. Hybridization was detected to an mRNA of similar size from oviductal tissue of the baboon, hamster, and mouse and to an mRNA of slightly smaller size in the rabbit, cow, and cat but not to any mRNA species from rat oviductal RNA. Slotblot analysis showed that the message was present in significantly greater (p < 0.05) concentrations in RNA from oviductal tissue from the late follicular stage than from the early follicular, early or late luteal, or postpartum stages. In conclusion, we have isolated the complete cDNA for a human oviductal glycoprotein. The presence of significantly greater amounts of the mRNA during the late follicular phase of the menstrual cycle is consistent with the proposed estrogen control. The mRNA for the oviductal glycoprotein is present only in the oviduct of an estrogen-treated baboon, and a cross-hybridizing RNA is found in oviductal RNA from various mammals.

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P D Jolly1, D J Tisdall1, Derek A. Heath1, S. Lun1, K. P. McNatty1 
TL;DR: Relationships between the occurrence and degree of granulosa cell apoptosis, cAMP response to FSH or LH, extant aromatase activity, and other previously established biochemical and morphometric indices of granULosa cell function and follicular atresia in this species are defined.
Abstract: Apoptosis is a process of selective cell deletion implicated as a mechanism underlying the process of ovarian follicular atresia. The aims of this study were 1) to test the hypothesis that granulosa cell death during follicular (> or = 4 mm diameter) atresia in cows occurs by apoptosis and 2) to define relationships between the occurrence and degree of granulosa cell apoptosis, cAMP response to FSH or LH, extant aromatase activity, and other previously established biochemical and morphometric indices of granulosa cell function and follicular atresia in this species. Granulosa cells and follicular fluid from individual follicles 4-18 mm in diameter were collected from luteal-phase cow ovaries. Follicles were classified by morphometric criteria as "healthy" (n = 45) or atretic (n = 34). Apoptosis in granulosa cells from each follicle was inferred from detection of internucleosomal DNA cleavage by 3'-end radiolabeling; it was quantitated both subjectively from intensity of oligonucleosome labeling (apoptosis [AP] score = 0, 1, 2, or 3) and objectively by beta-counting of low-molecular weight gel fragments (labeling index; LI). Extant aromatase activity (ng estradiol produced/10(6) cells/3 h) and cAMP response (pmol/10(6) cells) to different doses of FSH or LH (1-10,000 ng/ml) was determined for granulosa cells from most healthy follicles (n = 39). Apoptosis was detected in granulosa cells from all atretic follicles as well as from 76% of healthy follicles, from 80% (16 of 20) of follicles with follicular fluid estradiol to progesterone ratios > 1, and from 71% (10 of 14) of follicles with extant aromatase activity (> 2 ng/10(6) cells/3 h). For healthy and atretic follicles, degree of DNA fragmentation was inversely related to the number of granulosa cells recovered (as percentage maximum by follicle size). In healthy follicles, FSH stimulated cAMP synthesis is a dose-dependent manner in granulosa cells from all follicles examined (> or = 4 mm), but only 36% of these had appreciable aromatase activity. The cAMP response to FSH (per cell) increased with follicle size from 4-7 mm in diameter and remained high in granulosa cells from follicles > or = 8 mm with aromatase activity; in cells without aromatase activity, cAMP response to FSH decreased with increasing follicle size > or 8 mm. The cAMP response to LH was generally low or undetectable in granulosa cells from 4-8-mm follicles; it then increased linearly with increasing follicle diameter > or = 8 mm, but to a greater degree in cells with aromatase activity than in cells without.(ABSTRACT TRUNCATED AT 400 WORDS)

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TL;DR: The data indicate that the BSP proteins that are secretory products of the seminal vesicles bind to the sperm surface upon ejaculation, which is similar to previous work on choline phospholipids.
Abstract: Bovine seminal vesicles synthesize a family of closely related proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins). Recently, we showed that these proteins bind specifically to choline phospholipids. Since this class of phospholipids is the major phospholipid fraction of the spermatozoan membrane, we investigated the binding of BSP proteins to spermatozoa. Polyclonal antibodies against purified BSP proteins raised in rabbits were used to detect these antigens in bovine epididymal and ejaculated spermatozoa as well as in bovine seminal plasma. Comparison of spermatozoa taken from the caudae epididymides with ejaculated spermatozoa through use of various techniques, namely, surface labeling followed by immunoprecipitation and immunoblotting, showed that epididymal spermatozoa are devoid of BSP proteins whereas ejaculated spermatozoa possess membrane-bound BSP proteins. Through use of the indirect immunofluorescence technique, the ejaculated spermatozoa of bull were characterized by an immunoreaction restricted to the midpiece, acrosome, and postacrosomal region, but no specific immunostaining could be found on the surface of epididymal spermatozoa. Surface-labeled BSP proteins on spermatozoa could not be displaced with buffers containing high salt concentration (1 M NaCI), but could be displaced specifically with phosphorylcholine (alone or in combination with urea). The data indicate that the BSP proteins that are secretory products of the seminal vesicles bind to the sperm surface upon ejaculation.

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TL;DR: The disappearance of binding activities for IGFBP-2 and smaller-molecular-mass IGFBPs in E-A follicles suggests a possible regulatory role for IGFBBP in follicular maturation and on aromatase activity.
Abstract: The relationship between ovarian follicular steroidogenesis and insulin-like growth factor binding protein (IGFBP) activity was evaluated during the follicular phase of the bovine estrous cycle. In experiment 1, follicles were collected from cyclic cows (n = 11) slaughtered at 48 h after administration of prostaglandin F2 alpha (PGF; 35 mg i.m.). In experiment 2, cows were injected twice daily with saline (control) or FSH (FSH cows; total dosage = 42 mg) from Day 2 to Day 6 (estrus = Day 0) and with PGF (35 mg i.m.) on Day 7; follicles were collected from control cows (n = 20) slaughtered at 0, 24, 48, or 72 h and from FSH cows (n = 8) at 0 and 48 h after PGF. Follicular fluid was assayed for estradiol (E2), androstenedione (A4), progesterone (P4), and insulin-like growth factor-I (IGF-I) by RIA and for IGFBP activity by ligand blotting and densitometry. Intensities of the 34-kDa (IGFBP-2), 29-27-kDa, and 22-kDa IGFBP bands in follicular fluid were nondetectable or were lower (p or = 8 mm) E-active (E-A; E2 > 50 ng/ml and > P4) follicles than in large E-inactive (E-I), medium (5-7 mm), or small (< 5 mm) follicles. IGFBP-3 (44-40-kDa doublet) was unaffected by follicle stage in experiment 1, but IGFBP-3 was lower (p < 0.01) in follicular fluid of E-A vs. E-I large follicles in experiment 2. Profiles of IGFBP activity were similar in follicular fluid of small, medium, and E-I large follicles. In experiment 2, E2 concentrations in large E-A follicles increased (p < 0.01) from 0 to 48 h after the PGF injection for control cows but decreased (p < 0.01) for FSH cows, whereas follicular fluid IGFBP-2 binding activity decreased from 0 to 48 h after PGF in controls and increased in FSH cows (treatment x time, p < 0.05). IGFBP-3 binding was unaffected by FSH treatment or time after administration of PGF. Profiles of IGFBP activity in homogenates of granulosa or theca cells were similar to follicular fluid profiles except for the absence of IGFBP-3 binding activity. The disappearance of binding activities for IGFBP-2 and smaller-molecular-mass IGFBPs in E-A follicles suggests a possible regulatory role for IGFBPs in follicular maturation and on aromatase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

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TL;DR: Northern hybridization indicated that mRNA levels for clusterin and inhibin-beta B, Sertoli cell secretory proteins whose production normally increases during postnatal differentiation in vivo, were significantly increased by T3 or FSH alone.
Abstract: Transient neonatal hypothyroidism in the rat causes prolonged Sertoli cell proliferation, delayed Sertoli cell maturation, and increased adult Sertoli cell number, testis weight, and sperm production. Conversely, neonatal hyperthyroidism decreases Sertoli cell proliferation and ultimate testis size. This suggests that thyroid hormones might normally directly inhibit Sertoli cell proliferation while promoting maturation. However, these Sertoli cell effects could be due to secondary hormonal or metabolic effects of hypo- or hyperthyroidism. In this study, we directly tested thyroid hormone effects on Sertoli cell proliferation and differentiation in vitro. Sertoli cells from 5-day-old rat testes were grown in serum-free medium alone (controls) or with additional triiodothyronine (T3; 1-200 nM) and/or FSH (1 microgram/ml). After 4 days, cultures were used to obtain RNA for Northern hybridization or for thymidine autoradiography. Labeling index (LI) for control cultures and cultures receiving 100 nM T3 alone was 5.2 +/- 0.5% and 5.0 +/- 0.4%, respectively. The LI of FSH-treated cultures increased to 8.4 +/- 0.8% (p < 0.01 vs. control). Cultures treated with FSH and 1, 10, 100, or 200 nM T3 had LIs of 8.0 +/- 0.9%, 6.1 +/- 0.4%, 5.3 +/- 0.6%, and 4.8 +/- 0.6%, respectively; the last three values were less than for cells receiving FSH alone (p < 0.01) or FSH + 1 nM T3 (p < 0.05). Northern hybridization indicated that mRNA levels for clusterin and inhibin-beta B, Sertoli cell secretory proteins whose production normally increases during postnatal differentiation in vivo, were significantly increased by T3 or FSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

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TL;DR: The results indicate that the use of enucleated activated oocytes as cytoplasts for embryo reconstruction can increase the frequency of development to blastocyst of embryos reconstructed from unsynchronized donor blastomeres.
Abstract: The timing of pronuclear formation and the initiation and duration of the DNA synthetic period (S-phase) were determined during the first cell cycle of electrically activated ovine oocytes matured in vivo. Reconstructed embryos were produced by electro-fusion-mediated nuclear transfer of unsynchronized single blastomeres. These were derived from embryos produced in vivo at the 16-cell stage (Day 4) and transferred to enucleated metaphase II oocytes at the time of activation or to enucleated activated oocytes during early, mid, and late stages of the presumptive S-phase. The frequency of development to blastocyst was greatest in embryos reconstructed during the presumptive S-phase of enucleated activated oocytes than in embryos reconstructed at the time of activation (mean 55.4% vs. 21.3%). No significant differences were observed when embryos were reconstructed during early, mid, or late stages of the presumptive S-phase (61.3%, 45.7%, and 57.7%, respectively). The results indicate that the use of enucleated activated oocytes as cytoplasts for embryo reconstruction can increase the frequency of development to blastocyst of embryos reconstructed from unsynchronized donor blastomeres.

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TL;DR: Activated uterine hematopoietic cells are equipped to perform certain immunological and nonimmunological functions within their microenvironments that could have major influences on the course of pregnancy.
Abstract: Macrophages and a special subset of lymphocyte natural killer (NK) cells populate the uteri of cycling humans, mice, and rats. After implantation, major changes take place that have important functional implications. The macrophages and NK cells increase in number, are redistributed into specific uterine compartments, and exhibit markers consistent with cell activation. Activation enhances macrophage and NK cell production of a wide range of pleiotropic, multifunctional polypeptide growth factors, reactive oxygen intermediates, and bioactive lipids. Thus, activated uterine hematopoietic cells are equipped to perform certain immunological and nonimmunological functions within their microenvironments that could have major influences on the course of pregnancy.

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TL;DR: In this paper, the effects of sodium chloride (NaCl) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined.
Abstract: The effects of sodium chloride (NaCl) in Whitten's medium on intracellular glutathione concentration and on cytoplasmic maturation, as determined by monospermic penetration and male pronuclear formation of porcine oocytes, were examined. Porcine cumulus-oocyte complexes were cultured for 20 h in BSA-free Whitten's medium containing different NaCl concentrations (44.50, 68.49, 92.40, 116.40, or 140.35 mM) and supplemented with 10% porcine follicular fluid and hormonal supplements; the complexes were then cultured without hormonal supplements for an additional 20-h period. The mean width of the perivitelline space of oocytes was increased with decreased concentration of NaCl in the culture medium. Intracellular glutathione concentration was elevated in oocytes cultured in medium with lower NaCl concentrations. After co-culture with spermatozoa for 6 h and culture in modified Whitten's medium for an additional 6 h, there were no differences in maturation and penetration rates among experimental groups. However, the rate of male pronuclear formation was higher in oocytes matured in media with the lower NaCl concentrations. In addition, the rates of monospermic penetration and male pronuclear formation were higher in oocytes matured in medium containing 44.50 mM NaCl (59.3 +/- 8.1 and 70.9 +/- 2.0%, respectively) than in medium containing 68.49 mM NaCl (39.4 +/- 5.5 and 57.1 +/- 4.5%, respectively). These data indicated that decreasing NaCl concentration in maturation medium for porcine oocytes below the customary level improved the quality of the matured oocytes as reflected in higher intracellular glutathione content, wider perivitelline space, higher monospermic penetration rate, and increased frequency of male pronuclear formation.

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TL;DR: The present study suggests the functional homology of salmon GTH I and GTH II to mammalian FSH and LH, respectively, during gonadal development, by using in vitro ligand autoradiography of coho salmon gonads at various stages of sexual maturation.
Abstract: Receptors for two salmon gonadotropins, GTH I and GTH II, were localized by use of in vitro ligand autoradiography of coho salmon gonads at various stages of sexual maturation. The results in both sexes revealed the presence of two types of GTH receptors: type I (GTH-RI), which interacts with both GTHs, and type II (GTH-RII), which interacts specifically with GTH II. GTH-RI was found at all stages of spermatogenesis examined and was localized on cells that were most likely Sertoli cells; however, it could not be determined whether GTH-RI was also localized on Leydig cells. In contrast, GTH-RII was found only in Leydig cells in the testis from a spermiating fish. In the vitellogenic ovary, GTH-RI was localized in the thecal layer and intensely on granulosa cells; in the preovulatory follicle, in contrast, GTH-RI was found in the thecal layer and in interstitial connective tissue, but not in the granulosa layer. Among all the stages of oogenesis examined, only granulosa cells of the preovulatory follicle exhibited GTH-RII. The appearance of GTH-RII coincides well with the increase in plasma levels of GTH II that occurs during final oocyte maturation and spermiation in coho salmon. The nature, distribution, and timing of appearance of these two receptors can explain, at least in part, the results of previous studies on steroidogenic activities of the two GTHs. The present study also suggests the functional homology of salmon GTH I and GTH II to mammalian FSH and LH, respectively, during gonadal development.