Comparison of two culture systems for the in-vitro growth and maturation of mouse preantral follicles.

The objective of our study was to compare to ability of collagen-treated membranes and bovine collagen gels to maintain murine preantral follicle growth and development in-vitro. To fulfill that objective, murine follicle and oocyte growth rates were followed for ten days in culture. Meiotic competence and the capacity to reach the two-cell stage after in-vitro maturation and fertilization, respectively, were then assessed. We used preantral follicles from 12 day-old CF-1 female mice that were isolated by enzymatic digestion from ovaries. Follicles were placed either on collagen-treated membranes or embedded in a bovine collagen matrix. The follicles were grown, changing the media and obtaining measurements every other day for ten days. Following culture, the granulosa-oocyte complexes were matured; the resultant metaphase II arrested oocytes were inseminated and cultured to the two-cell stage. The data was analyzed with significance considered for probability values of p < 0.05. We performed individual measurements on 650 follicles in seven separate experiments. Forty-eight hours after initial seeding and throughout the entire length of culture, both the follicles and oocytes grown in the collagen matrix were larger than follicles cultured on collagen-treated membranes (p < .0001). However, oocyte recovery rates were higher among follicles cultured on collagen-treated membranes (p < .01). Similar percentages of meiotically competent oocytes, fertilization and cleavage rates were observed in both groups. Our results show that mouse preantral follicles display a greater growth rate when grown embedded in a collagen gel matrix. This may be due to the maintenance of a normal three-dimensional organization of the follicle within the collagen matrix. However, this system does not enhance meiotic competency or fertilization rates in the mouse when compared to culture on collagen-treated membranes.

Analysis of the human zona pellucida during culture: correlation with diagnosis and the preovulatory hormonal environment.

PURPOSE: The objective of this study was to analyze sequentially the human zona pellucida changes in an in vitro fertilization program as it relates to several variables. METHODS: The zona pellucida thickness was measured daily in zygotes and cleavage-stage embryos on a Nikon inverted microscope equipped with Hoffman modulation contrast optics, using an ocular micrometer. A total of 512 embryos from 96 patients was evaluated. RESULTS: There was a highly significant direct correlation between zona thickness and preovulatory estradiol and basal day 3 FSH levels (P < 0.02 and P < 0.0006, respectively). This relationship showed a rapid reversal following 48 hr of culture; embryos from patients with the highest FSH levels had thinner zonae prior to transfer (P < 0.0007). The zonae from patients with unexplained infertility were thicker (19.4 +/- 2.7 microns) than those from patients with endometriosis (17.7 +/- 2.2 microns), tubal (17.5 +/- 2.4 microns), or male-factor infertility (16.4 +/- 2.7 microns) (P < 0.0001) on the first day of culture. CONCLUSIONS: We hypothesize that the thickness of the human zona pellucida is influenced by the preovulatory hormonal environment and diagnosis. These factors should be considered as part of the embryo quality evaluation prior to transfer or when assessing the possibility of using assisted hatching. More studies are needed to understand the factors regulating the thickness of the human zona pellucida.

Micromachined analytical devices: microchips for semen testing.

Micromachined devices (microchips) have been designed and tested for a range of clinically important assays. In this study we compare sperm motility determined using disposable glass microchips and a conventional Makler chamber. The 17 x 14 mm glass microchips contained three etched test structures each comprising either duplicate or quadruplicate analytical microchannels. Semen samples with sperm counts ranging from 21 to 78 million sperm per ml and forward progression scores of from 1+ to 3+ were evaluated and swimming times ranging from 360 s (3.3+ progression) to 770 s (1+,2 forward progression) observed in the microchips. Motility determined by the time taken for sperm to swim to the end of a microchannel (100 microns wide x 40 microns deep x 10 mm long) in the microchip correlated with forward progression of the sperm determined by the conventional Makler chamber method. This study demonstrates the feasibility of microchips for sperm motility testing and suggests that this technique would be applicable to the study of other types of motile cells.

Sequential analysis of zona thickness during in vitro culture of human zygotes: correlation with embryo quality, age, and implantation.

Zona pellucida thickness was measured daily in zygotes and cleavage stage embryos. Measurements were performed on a Nikon inverted microscope equipped with Hoffman modulation optics, using an ocular micrometer. Zona thickness of each zygote/embryo was measured four times, the zygote/embryo was then "rolled over," and four more measurements were repeated for a total of eight. The zygotes/embryos were photographed daily and the measurements repeated on the prints. Subsequently, the mean zona thickness for each stage was calculated. A total of 81 patients (mean age 33.8 +/- 4.2) participated in the study. A total of 427 embryos were evaluated. Categorical data differences between groups were evaluated by ANOVA and multiple linear regression. For nominal data, the Kruskal-Wallis test was applied; when P < 0.05 the differences were considered to be significant. We found that the average zona thickness on day 1 of in vitro culture was 17.7 +/- 0.14 microns; 16.3 +/- 0.14 microns on day 2 and 14.9 +/- 0.14 microns on day 3 (P < .0001). When the zona thickness was analyzed in relation to the number of blastomeres on day 3 of culture, there was a highly significant correlation with blastomere number (P < .0001). Similarly, there was a highly significant correlation with embryo grade (P < .005) and fragmentation (P < .001). The data were also analyzed for embryos transferred that resulted in a successful pregnancy, revealing that embryos in a pregnancy cycle had significantly thinner zonae pellucidae (P < .0001), when compared to embryos that were not transferred or from nonconceptual cycles. The average zona thickness also decreased with age, and was most apparent after 35 years. Changes in zona thickness correlated with the number of blastomeres, grade, fragmentation, age and were more evident in embryos transferred from cycles resulting in successful pregnancies. Therefore, zona pellucida measurements should be included in the overall assessment of embryo quality, since this information may be useful in the selection of optimal embryos for transfer.

Growth factors in preimplantation development: role of insulin and insulin-like growth factors.

In vitro studies of preimplantation embryos from a number of mammalian species have shown that the oviduct and uterus contain growth factors that stimulate cellular proliferation and differentiation of preimplantation embryos. The mammalian preimplantation embryo was first viewed as an autonomous entity, due to the ease with which mouse embryos could be cultured from the two-cell stage on to the blastocyst. This view changed following studies in other species, notably domestic animals, which revealed the presence of 'blocks' to development, when embryos were cultured in vitro. Another line of evidence leading to the view that the maternal environment is crucial for optimal development, was the finding that embryos cultured in vitro lag developmentally behind their in vivo counterparts. This developmental retardation could be ameliorated when washings from the reproductive tract or specific growth factors were added to the media. The expression of genes for growth factors and their receptors is regulated in a tissue-specific manner as well as temporally and spatially during mammalian development. In this review, information regarding the expression and role of polypeptide growth factors of the insulin family during preimplantation mammalian development is summarized.

Classification of male factor infertility relevant to in-vitro fertilization insemination strategies using mannose ligands, acrosome status and anti-cytoskeletal antibodies.

Polyvalent mannose ligands in the presence of free mannose act as zona pellucida agonists which rapidly induce acrosome exocytosis in competent motile human sperm from fertile donors following in-vitro capacitation. Quantification of the binding patterns of fluorescein isothiocyanate-labelled mannosylated albumins and of specific antisera which recognize mannose receptors and other related integral sperm membrane proteins as well as the incidence of induced acrosome exocytosis after capacitation has allowed us to identify three categories of male infertility. Category 1 males have normozoospermic semen parameters, their spermatozoa have elevated sperm cholesterol values and fail to fertilize oocytes in vitro after standard short-term incubations. These spermatozoa do not bind mannose ligands and do not show spontaneous or induced acrosome reactions, but treatments to remove cholesterol from the spermatozoa (e.g. prolonged incubation in the presence of sterol acceptors) confer the ability to fertilize. Cholesterol loading and unloading experiments have demonstrated the reversible character of sperm membrane properties in category 1 male infertility. Category 2 males have normal-appearing spermatozoa in semen which express mannose ligand receptors on incubation, but fail to undergo acrosome reactions in response to mannose treatment. Interestingly, all category 2 males identified in this study have clinical varicocele. Category 3 males have semen which may be normozoospermic or teratozoospermic with, in some cases, high percentages of tapering spermatozoa in the absence of clinical varicocele. Spermatozoa from category 3 men are deficient in a superfamily of integral membrane proteins whose cytoplasmic tails have myosin motors as identified by amino acid sequence analysis and anti-myosin antibody reactivity. Their spermatozoa do not express mannose ligand receptors or undergo induced acrosome reactions. Fertilization with category 2 and 3 semen is only achieved by micromanipulation procedures. These findings illustrate the practical application of basic research for infertility classification.

A comparison of three methods for detecting the acrosome reaction in human spermatozoa.

This study was designed to compare three different fluorescent probes to assay the acrosome reaction in human spermatozoa: chlortetracycline (CTC), mannosylated bovine serum albumin (BSA) labelled with fluorescein (MAF), and quinacrine (QN). Normal human sperm ejaculates were washed and allowed to swim up for 30-60 min. Samples were examined under epifluorescence for the percentage of the acrosome reacted spermatozoa, as detected by the three probes. There was no significant differences between samples of fresh, uncapacitated spermatozoa evaluated with CTC, MAF or QN; all gave < 10% reacted. Following capacitation for 3 h, the percentage of spontaneously reacted spermatozoa was higher than in fresh spermatozoa; CTC and MAF gave the same percentage (12%), while QN indicated a higher percentage (18%) of reacted spermatozoa (P < 0.001). Following exposure to ionophore A23187 at 1 h, the percentage of acrosome reactions increased to a mean of 31% as detected with CTC or MAF; the mean percentage (45%) was significantly higher with QN (P < 0.0001). Further incubation up to 2 h with A23187 did not change these percentages. These results suggest that the QN probe detects the onset stage of the acrosome reaction, whereas the CTC and MAF probes detect the later stages in which the acrosomal cap is lost. Use of the two types of probe provides a means for finer resolution of the time course of the acrosome reaction in the human spermatozoa.

Post-transcriptional stimulation of transforming growth factor beta 1 mRNA by TGF-beta 1 treatment of transformed human osteoblasts.

Following exogenous administration of transforming growth factor-beta 1 (TGF-beta 1) polypeptide to the human osteosarcoma cell line TE-85, we observed a 2- to 6-fold stimulation of steady-state TGF-beta 1 mRNA. The stimulation was dose- and time-dependent, as judged from Northern blot hybridization analyses. A 2- to 6-fold increase of the TGF-beta 1 polypeptide was also found in the media of these cells after TGF-beta 1 treatments. The autostimulation of TGF-beta 1 mRNA was nullified by cycloheximide treatment of the cells. The in vitro transcription rates of the TGF-beta 1 gene by isolated nuclei were not altered by TGF-beta 1 treatment. Under conditions of transcriptional inhibition, the stability of TGF-beta 1 mRNA was enhanced nearly two-fold by TGF-beta 1 treatment. Our findings indicate that TGF-beta 1 can stimulate autologous gene expression and subsequent polypeptide translation by a post-transcriptional mechanism requiring protein synthesis in human osteoblast-like cells. The recognized versatility of TGF-beta 1 autostimulation mechanisms (transcriptional and post-transcriptional) in other mesenchymal cells may apply also to skeletal cells, further underscoring the broad and potent activities of this cytokine.

Mouse embryonic stem cells express receptors of the insulin family of growth factors.

Insulin and insulin-like growth factors (IGF-I and -II) are members of a family of growth factors which are known to be developmentally regulated during preimplantation mouse embryogenesis. The physiological actions of the insulin family of growth factors are mediated by interactions with specific cell surface receptors that are detectable on the cells of preimplantation mouse embryos. Mouse embryonic stem (ES) cells are totipotent cells derived directly from the inner cell mass of the blastocyst. ES cells have the ability to differentiate into all three germ layers and have unlimited growth potential under certain culture conditions. The great advantage of ES cells is the ability to obtain large amounts of tissue for biochemical studies as compared with preimplantation embryos. To examine in greater detail the biological actions of the insulin family of growth factors, the expression of their cognate receptors on ES cells was examined. ES cells were cultured in DMEM medium supplemented with leukemia inhibitory factor (LIF) to maintain the undifferentiated state. Receptor expression was evaluated at the mRNA level using the reverse transcription polymerase chain reaction (RT-PCR), and at the protein level by radioactive labeled ligand-receptor binding assay. Using RT-PCR, mRNAs of all three growth factor receptors were detected in ES cells. Messenger RNA from ES cells was reverse transcribed into cDNA by AMV reverse transcriptase at 42 degrees C for 1 hr. The reverse transcription reaction was amplified with Taq polymerase and specific primers for insulin, IGF-I, or IGF-II receptors by PCR. RT-PCR and the control plasmid cDNA PCR products were resolved electrophoretically on 3% agarose gels. Each amplified PCR product showed the predicted correct size.(ABSTRACT TRUNCATED AT 250 WORDS)

Can the immunobead assay for detecting sperm antibodies in fresh samples be reproduced in cryopreserved/thawed human spermatozoa?

PROBLEM: To evaluate the reproducibility of the Immunobead Assay (IBA) on sperm samples before and after cryopreservation. METHOD: Sperm samples (fresh and post-thaw) from known antibody negative donors (N = 20) were evaluated for percent immunobead binding by IBA following incubation with known antibody-positive serum. RESULTS: In both fresh and thawed negative samples, the mean sperm head binding was 0.5% +/- 0.5, the mean sperm tail binding was 2.0% +/- 2.0 and the mean sperm head-tail binding was 3.0% +/- 2.0 for IgG, IgA and IgM type antibodies, respectively. The same samples exposed to positive sera showed 40.0% +/- 10.0 mean head binding, 7.0% +/- 8.0 mean tail binding and 47.0% +/- 11.0 mean head-tail binding. CONCLUSIONS: IBA is highly reproducible for detecting sperm antibodies in both fresh and cryopreserved/thawed samples of human spermatozoa.

Insulin family growth factors have specific effects on protein synthesis in preimplantation mouse embryos.

Previously constructed protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage, have been used to analyze the effects of insulin, IGF-I, and IGF-II on protein synthesis in these developmental stages. Proteins were labeled by placing, for 2 hr, synchronous cohorts of 35-50 embryos into human tubal fluid (HTF) medium containing L-[35S]-methionine (1 mCi/ml) in the presence or absence of one of the growth factors. The embryos were then washed with medium and lysed. Samples were processed for 2-D gel analysis. For each embryonic stage and each growth factor, four or five experimental replicates were done and the gel images were compared using the PDQUEST system. Using the computer-assisted analysis, we were able to identify proteins that showed a statistically significant (P < 0.05) change in synthesis. At the eight-cell stage of development insulin caused increased synthesis of two proteins and decreased synthesis in three proteins. Insulin-treated blastocyst stage embryos exhibited an increased synthesis in eight proteins and decreased synthesis for one protein. The effect of IGF-I at the eight-cell stage of development was mostly inhibitory; the synthesis of only one protein increased and the synthesis of five proteins showed a decrease. Similar results were obtained with blastocyst stage embryos; four proteins demonstrated an increase in synthesis while 14 proteins showed a decrease. Eight-cell stage embryos incubated with IGF-II had seven proteins with a decreased synthesis, although in blastocyst stage embryos, nine proteins showed increased synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein databases for compacted eight-cell and blastocyst-stage mouse embryos.

High-resolution two-dimensional sodium dodecyl sulfate-polyacrylamide (2D-SDS) gel electrophoresis combined with computerized analysis of gel images was used to construct and analyze protein data-bases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage. These stages were chosen for their ease in identification of multiple synchronous embryos. Synchronous cohorts of 30-50 embryos were labelled with L-[35S]methionine for 2 hr. The embryos were then lysed in 30 microliters hot SDS sample buffer, and the lysates were stored at -80 degrees C until the gels were run. Five replicates were run for eight-cell embryos, and four for blastocyst-stage embryos. The samples were processed for 2D gel electrophoresis and fluorography; multiple exposures were made. Gel images were analyzed using the PDQUEST system, and databases were constructed. Analysis of the databases for both developmental stages showed high reproducibility of protein spots in multiple gel images. Of 1,674 total spots in eight-cell embryo standards, > 79% of spots had a percentage error (S.E.M./average) < 50%, and > 45% had a percentage error < 30%. Similarly, of 1,653 total spots in blastocyst-stage embryo standards, 74% of spots had a percentage error < 50%, and approximately 47% of spots had a percentage error < 30%. Forty-three spots (approximately 3% of the total spots) were found to be detected only in the eight-cell stage, while 75 spots were detected solely in the blastocyst stage. Sixty-nine proteins showed a greater than threefold increase in isotope incorporation from the eight-cell to the blastocyst stage, with a percentage error < 50% in both the eight-cell and the blastocyst stages. In contrast, 41 of the proteins showed a decrease during this period. Analysis of the protein databases described in this study has allowed us to document the overall quantitative changes in proteins from the compacted eight-cell stage to the blastocyst stage of mouse preimplantation development. These data-bases provide a valuable tool for further detailed quantitative analysis of specific proteins associated with developmental events. In addition they will permit analysis of the effects of environmental factors, such as growth factors, on early embryo development.

Applications of a microfabricated device for evaluating sperm function.

Mesoscale structures (microns dimensions, nL-pL volumes) have been designed and fabricated in silicon for use in various analytical tasks. We studied sperm motility and performed sperm selection in channels (80 microns wide x 20 microns deep), branching structures (40 microns wide x 20 microns deep, eight bifurcations), and channels containing barriers (7 microns feature size). Sperm-cervical mucus and sperm-hyaluronic acid interactions were assessed by using appropriate microchannel-chamber structures filled with either cervical mucus or hyaluronic acid. Simultaneous assessment of the potency of different spermicides (e.g., nonoxynol-9, C13G) and spermicide concentrations was achieved with structures comprising chambers containing spermicide connected via channels to a central chamber into which semen was introduced. Semen was also tested for the presence of sperm-specific antibodies by using microchannels filled with human anti-IgG antibody-coated microbeads.

Functions of the IGFs in early mammalian development.

The identification of growth factors and/or receptors produced by mammalian embryos or present in the maternal reproductive tract is of basic interest, as well as having practical application. Early studies established that receptors binding insulin and the insulin-like growth factors (IGFs) are expressed by preimplantation mouse embryos. These studies have been confirmed at the molecular level using RT-PCR techniques. In addition, high resolution electron microscopy has shown that insulin is internalized by the cells of the blastocyst stage mouse embryo, and that immunologically intact insulin is detectable in the cells of the trophectoderm and inner cell mass. Similar studies with gold labelled IGF-I have shown that this ligand is also bound and internalized by mouse blastocysts. However, although all blastocysts express receptors that bind IGF-I on the basolateral cell surface of the trophectoderm, only 30% exhibit apically located receptors. In order to elucidate the functions of IGFs in early mouse development, we are in the process of constructing protein data-bases for embryos at the eight-cell and blastocyst stage. By the use of the database, it should prove possible to elucidate targets of growth factor action.

Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime.

While washing of human sperm cells by centrifugation and resuspension is a procedure in widespread use, there have been indications that this procedure per se may be harmful to the cells. The objective of this study was to investigate this question. To this end, a method for the clean separation of motile human spermatozoa from seminal plasma in the absence of centrifugation was developed, using a modified swim-up procedure, in which liquefied semen was mixed with an equal volume of 30 mg/ml dextran in medium, and the mixture overlaid with medium containing 5 mg/ml bovine serum albumin, forming two discreet layers with stable interface. The percentage of motile cells in a given sample was consistently > 80% immediately after recovery. Damage to the cells was assessed by loss of motile cells during incubation up to 96 h post-recovery. Comparison of aliquots of spermatozoa obtained by the dextran swim-up procedure showed that the aliquot subjected to centrifugation had 4 +/- 3% motile cells after 48 h, while the untreated aliquot had 52 +/- 12%. The aliquots showed no difference 1 h post-recovery. Similar results were obtained with spermatozoa that had been centrifuged in seminal plasma and resuspended in fresh plasma, then recovered by dextran swim-up. The delayed onset of motility loss in the centrifuged samples implies that this treatment induces sublethal damage in the cells. Comparison of the standard swim-up and Percoll gradient methods for sperm recovery, both of which involve centrifugation steps, showed decline in motility of the samples similar to that seen with dextran swim-up of centrifuged cells. We conclude that centrifugation per se induces sublethal damage in human spermatozoa, independently of treatment method, and suggest that recovery methods for human spermatozoa which avoid centrifugation might partially alleviate the damage incurred by these cells during cryopreservation.

Mouse preimplantation embryos exhibit receptor-mediated binding and transcytosis of maternal insulin-like growth factor I.

High-resolution microscopy in conjunction with colloidal gold-labeled insulin-like growth factor I (IGF-I) has been used to provide evidence that the IGF-I receptor is first detected in 8-cell-stage mouse embryos, confirming the results of previous reverse transcriptase polymerase chain reaction (RT-PCR) studies. Specificity for the IGF-I receptor was demonstrated by displacement with unlabeled IGF-I and dual-labeling experiments with colloidal gold-labeled or unlabeled insulin. Labeled IGF-I ligand is internalized by means of receptor-mediated endocytosis following its concentration in coated pits, and it can be visualized within cytoplasmic organelles. Immunocytochemical analyses at the blastocyst stage, using gold-labeled antibodies to the receptor, confirmed the expression of IGF-I receptors on all cells of the embryo. Similar studies with antibodies directed against the ligand demonstrated that IGF-I internalized by the embryo in vivo is maternally derived. Approximately 40% of blastocysts showed apical plasma membrane binding of gold-labeled ligand ("responders"), while approximately 60% did not demonstrate binding ("nonresponders"); however, both classes of embryo expressed receptors on basolateral membranes of trophectoderm cells and on the surface of inner masses. Functional studies show that incubating embryos in physiological levels of IGF-I (40 ng/ml) results in increased numbers of cells in the inner cell mass (p < 0.05), but not the trophectoderm, as compared to controls.

Growth factors in preimplantation mammalian embryos.

It has been hypothesized for some time that secretions of the oviduct and uterus are involved in stimulating cell proliferation in preimplantation mammalian embryos and promotion of early differentiation events that lead to successful implantation. At least some of the regulatory factors present within uterine secretions are growth factors that can act along a paracrine pathway by binding to specific receptors on embryonic cells. A list of polypeptide growth factors present in uterine tissues and fluids has been previously published by Brigstock et al. (1989) and along with those reported in this review, includes EGF, TGF-alpha, insulin, IGF-I, IGF-II, IGF-BPs, acidic and basic FGF, and CSF-1. The early embryo itself produces a number of growth factors and receptors. A summary of those covered in this review, including temporal aspects of their expression, is contained in Table 2.1. Most of the data are from studies on mouse embryos but where, possible, we have also included reports for other mammalian embryos. Taken together with the factors present in oviduct and uterine secretions, it is clear that preimplantation embryos reside in an environmental milieu in which they are exposed to growth factors of many kinds and that regulatory pathways at the autocrine, juxtacrine, and paracrine levels may all be operating. From a functional point of view, many of the factors we have reviewed have been shown to be able to enhance development when added to medium for culture of preimplantation embryos. The exact circuit or pathway and mechanism through which they exert their effects remain, for the most part, to be elucidated. None the less, a number of general features regarding growth factor function during preimplantation development have emerged. There appears to be a redundancy of gene products within several growth factor families, all of which can stimulate cell proliferative or metabolic events when added exogenously to preimplantation embryos in culture. Perhaps, then, in addition to functions of growth factors acting singly on their specific receptors, combinations of factors are important for induction of a specific developmental response. We have included many examples of synergistic actions of growth factors during preimplantation development in the previous sections. It is also possible that the result of combinations of factors may involve a process of interference whereby exposure of embryonic cells to one growth factor may compromise its ability to bind and respond to another.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene expression in pre-implantation mammalian embryos.

The pre-implantation mammalian embryo is initially under the control of maternal informational macromolecules that are accumulated during oogenesis. Subsequently, the genetic program of development becomes dependent upon new transcription derived from activation of the embryonic genome. Several embryonic transcripts including those that encode growth factors, cell junction components and plasma membrane ion transporters are required for normal progression of the embryo to the blastocyst stage. The pattern of genes expressed and the overall program of development is subject to the influences of genomic imprinting as well as external influences encountered by the embryo within the maternal reproductive tract.