Automated Measurements of Key Morphological Features of Human Embryos for IVF.

A major challenge in clinical In-Vitro Fertilization (IVF) is selecting the highest quality embryo to transfer to the patient in the hopes of achieving a pregnancy. Time-lapse microscopy provides clinicians with a wealth of information for selecting embryos. However, the resulting movies of embryos are currently analyzed manually, which is time consuming and subjective. Here, we automate feature extraction of time-lapse microscopy of human embryos with a machine-learning pipeline of five convolutional neural networks (CNNs). Our pipeline consists of (1) semantic segmentation of the regions of the embryo, (2) regression predictions of fragment severity, (3) classification of the developmental stage, and object instance segmentation of (4) cells and (5) pronuclei. Our approach greatly speeds up the measurement of quantitative, biologically relevant features that may aid in embryo selection.

Optimal timing for blastomere biopsy of 8-cell embryos for preimplantation genetic diagnosis.

STUDY QUESTION: What is the optimal timing for blastomere biopsy during the 8-cell stage, at which embryos will have the best implantation potential? SUMMARY ANSWER: Fast-cleaving embryos that are biopsied during the last quarter (Q4) of the 8-cell stage and are less affected by the biopsy procedure, and their implantation potential is better than that of embryos biopsied earlier during the 8-cell stage (Q1-Q3). WHAT IS KNOWN ALREADY: Blastomer biopsy from cleavage-stage embryos is usually performed on the morning of Day 3 when the embryos are at the 6- to 8-cell stage and is still the preferred biopsy method for preimplantation genetic diagnosis (PGD) for monogentic disorders or chromosomal translocations. Human embryos usually remain at the 8-cell stage for a relatively long 'arrest phase' in which cells grow, duplicate their DNA and synthesize various proteins in preparation for the subsequent division. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study. The study group (195 embryos) included all 8-cell stage embryos that underwent blastomere biopsy for PGD for monogenetic disorders and chromosomal translocations in our unit between 2012-2014 and cultured in the EmbryoScope until transfer. The control group (115 embryos) included all embryos that underwent intracytoplasmic sperm injection without a biopsy during the same period. PARTICIPANTS/MATERIALS, SETTING, METHODS: The 8-cell stage was divided into four quarters: the first 5 h post-t8 (Q1), 5-10 h post-t8 (Q2), 10-15 h post-t8 (Q3) and at 15-20 h post-t8 (Q4). Non-biopsied control embryos were divided into four equivalent quarters. Embryos were evaluated for timing of developmental events following biopsy including timing of first cleavge after biopsy, timing of comapction (tM) and start of blastulation (tSB). Timing of these events were compared between PGD and control embryos, as well as with 56 PGD implanted embryos with Known Implantation Data (PGD-KID-positive embryos). MAIN RESULTS AND THE ROLE OF CHANCE: Embryos that were biopsied during Q3 (10-15 h from entry into 8-cell stage) were delayed in all three subsequent developmental events, including first cleavage after biopsy, compaction and start of blastulation. In contrast, these events occurred exactly at the same time as in the control group, in embryos that were biopsied during Q1, Q2 or Q4 of the 8-cell stage. The results show also that embryos that were biopsied during Q1, Q2 or Q3 of the 8-cell stage demonstrated a significant delay from the biopsied implanted embryos already in t8 as well as in tM and tSB. However, embryos that were biopsied during Q4 demonstrated dynamics similar to those of the biopsied implanted embryos in t8 and tM, and a delay was noticed only in the last stage of tSB. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study that is limited to the timing of biopsy that is routinely performed in the IVF lab. A prospective study in which biopsy will be performed at a desired timing is needed in order to differ between the effect of biopsy itself and the cleavage rate of the embryo. WIDER IMPLICATIONS OF THE FINDINGS: Our findings showed that blastomere biopsy can be less harmful to further development if it is carried out during a critical period of embryonic growth, i.e during Q4 of the 8-cell stage. They also demonstrated the added value of time-lapse microscopy for determining the optimal timing for blastomere biopsy. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the routine budget of our IVF unit. TRIAL REGISTRATION NUMBER: N/A.

Human embryonic stem cells carrying an unbalanced translocation demonstrate impaired differentiation into trophoblasts: an in vitro model of human implantation failure.

Carriers of the balanced translocation t(11;22), the most common reciprocal translocation in humans, are at high risk of creating gametes with unbalanced translocation, leading to repeated miscarriages. Current research models for studying translocated embryos and the biological basis for their implantation failure are limited. The aim of this study was to elucidate whether human embryonic stem cells (hESCs) carrying the unbalanced chromosomal translocation t(11;22) can provide an explanation for repeated miscarriages of unbalanced translocated embryos. Fluorescent in situ hybridization and karyotype analysis were performed to analyze the t(11;22) in embryos during PGD and in the derived hESC line. The hESC line was characterized by RT-PCR and FACS analysis for pluripotent markers. Directed differentiation to trophoblasts was carried out by bone morphogenetic protein 4 (BMP4). Trophoblast development was analyzed by measuring ß-hCG secretion, by ß-hCG immunostaining and by gene expression of trophoblastic markers. We derived the first hESC line carrying unbalanced t(11;22), which showed the typical morphological and molecular characteristics of a hESC line. Control hESCs differentiated into trophoblasts secreted increasing levels of ß-hCG and concomitantly expressed the trophoblast genes, CDX2, TP63, KRT7, ERVW1, CGA, GCM1, KLF4 and PPARG. In contrast, differentiated translocated hESCs displayed reduced and delayed secretion of ß-hCG concomitant with impaired expression of the trophoblastic genes. The reduced activation of trophoblastic genes may be responsible for the impaired trophoblastic differentiation in t(11;22)-hESCs, associated with implantation failure in unbalanced t(11;22) embryos. Our t(11;22) hESCs are presented as a valuable human model for studying the mechanisms underlying implantation failure.

PGD-derived human embryonic stem cell lines as a powerful tool for the study of human genetic disorders.

Human embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocyst embryos. They are established from spare embryos that have been obtained by in vitro fertilization (IVF) and donated for research purposes. The ICM-derived cell lines have two unique properties, they can be propagated indefinitely in culture and have the potential to develop into practically any cell type in vitro and in vivo. Human embryonic stem (hES) cells carrying specific mutations can be used as a valuable tool for studying genetic disorders in human. One favorable approach to obtain such mutant ES cell lines is their derivation from affected preimplantation genetic diagnosed (PGD) embryos. This review focuses on the importance of deriving human ES cell lines from genetically abnormal embryos, especially in cases where no good cellular and/or animal models exist.

Probability of sperm detection in nonobstructive azoospermic men undergoing testicular sperm extraction procedures unrelated to clinical parameters.

The study was conducted to evaluate the significance of preoperative clinical parameters for detection of mature testicular sperm cells in nonobstructive azoospermic men. Sixty-five consecutive men with nonobstructive azoospermia underwent testicular sperm extraction procedures. Testicular samples were analyzed histologically with patterns classified as mature spermatogenesis (normal or partial), arrest of spermatogenesis, and Sertoli cell only. Testicular sperm cells were isolated for use in an IVF/ICSI program. Histologic patterns and detection rate of sperm cells were correlated to clinical characteristics. Mature sperm cells were found in all levels of serum FSH. The men were divided into 3 groups based on their clinical characteristics (serum FSH level and testicular size). The distribution of the different testicular histologic patterns, as well as detection rate of sperm cells, was similar in all groups. No correlation was found between serum levels of FSH, LH, prolactin, or testosterone and sperm presence. None of these parameters, nor the testicular size and consistency, can serve as predictive variables of the histological pattern or the presence of mature sperm cells in the testicular biopsies in cases of nonobstructive azoospermia. Until an effective predictive tool is available, a trial of sperm retrieval is recommended for all azoospermic men independent of their clinical characteristics.

Vascularity index distribution within the testis: a technique for guiding testicular sperm extraction.

Azoospermia is defined as the absence of spermatozoa in the ejaculate, although some foci of spermatogenesis may exist in the testes of these men. Currently, there are no clinical, seminal or hormonal parameters for identifying spermatogenesis within the testis sufficient for achieving genetic offspring. As a result, multiple biopsies are performed at several arbitrary sites of both testes in search of spermatozoa. We developed a power Doppler (PD) ultrasound (US) image-based technique that predicts sites with the greatest potential for spermatogenesis. PDUS images of the testes of azoospermic men were acquired at seven cross-sections to reconstruct a 3-D matrix for constructing a spatial map of preferential regions where spermatozoa are most likely to exist. This technique may obviate the need for arbitrary multiple biopsies that inflict some degree of damage upon testicular tissue, and may increase the success rate of identifying viable spermatozoa in testicular biopsies.

Oocyte activation: lessons from human infertility.

During fertilization, the spermatozoon penetrates through the cumulus cells and the zona pellucida that surrounds the oocyte, before it binds and fuses with the oocyte plasma membrane to induce activation. In vitro fertilization (IVF) studies performed in non-human mammals have contributed extensive knowledge regarding the mechanisms by which the spermatozoon activates the meiotic-arrested oocyte to resume meiosis, cleave and develop into an embryo. Although IVF has been used extensively for treating subfertile couples, not all of them were able to benefit from this procedure. In intracytoplasmic sperm injection (ICSI), one viable spermatozoon only is sufficient for successful fertilization of a single oocyte. Moreover, the injected fertilizing spermatozoon bypasses several physiological barriers, compared with IVF, which together could explain the high success rate for this procedure. ICSI has also allowed the identification of sperm components that are required for successful fertilization.

Increasing synthetic serum substitute (SSS) concentrations in P1 glucose/phosphate-free medium improves implantation rate: a comparative study.

PURPOSE: To assess the comparative efficacy of IVF medium (MediCult, with 5.2 mM glucose) and a glucose/phosphate-free medium, P1 (Irvine Scientific), and to investigate the influence of increasing the serum supplementation (synthetic serum substitute; SSS; Irvine Scientific) to P1 on embryo development and implantation. METHODS: Patients were randomly assigned to IVF medium (Group 1, cycles n = 172) or P1 supplemented with 10% SSS (Group 2, cycles n = 229) according to the medium scheduled for use on the day of oocyte retrieval. Another 555 IVF consequent cycles (Group 3) were performed using increased SSS concentrations (20%) in P1 medium. RESULTS AND CONCLUSION: In this large series of IVF cycles, we herein demonstrate that significantly higher pregnancy and implantation rates were found when embryos were cultured in glucose/phosphate-free medium P1 supplemented with 20% SSS compared to supplementation with the lower SSS concentration and with IVF medium.

Testicular sperm retrieval and cryopreservation prior to initiating ovarian stimulation as the first line approach in patients with non-obstructive azoospermia.

The potency for fertilization and successful implantation was compared between fresh and cryopreserved testicular spermatozoa obtained from the same patient with non-obstructive azoospermia. Spermatozoa cryopreserved at the outset were also evaluated. Non-obstructive azoospermic men (n = 55) underwent testicular sperm extraction (TESE); mature spermatozoa were found in 33 (60%) of them. Of 57 intracytoplasmic sperm injection (ICSI) cycles in 25 patients, 15 used fresh spermatozoa (14 patients, group 1), 24 used the excess spermatozoa cryopreserved after 'fresh' ICSI (11 couples who did not conceive in the 'fresh' cycle, group 2) and 18 cycles used cryopreserved spermatozoa at the outset (11 other patients, group 3). Fertilization, cleavage, embryo quality, implantation and take home baby rates were not significantly different in groups 1 and 2, and 6/14 couples ultimately had healthy babies (42.8% cumulative take home baby rate per TESE). In group 3, neither the fertilization rate, embryo development, pregnancy nor implantation rates per embryo transfer were significantly different from groups 1 and 2. The cumulative delivery and ongoing pregnancy rate in this group was 36. 4%. Cryopreservation did not impair the availability of motile spermatozoa for ICSI. When immotile spermatozoa were injected, however, fertilization rate decreased dramatically. Since criteria for predicting the presence of spermatozoa in the testicular tissue of patients with non-obstructive azoospermia are inadequate, it is suggested that TESE be performed prior to initiating ovarian stimulation.

Multiple testicular sampling in non-obstructive azoospermia--is it necessary?

Spermatogenesis may be focal in non-obstructive azoospermia. The present study was conducted to determine whether the performance of multiple, rather than a single testicular sample contributes to obtaining spermatozoa in amounts sufficient for fertilization and cryopreservation in non-obstructive, azoospermic patients. Furthermore, the aim was to clarify the significance of location for retrieval from the testis in such cases. Three biopsies were taken from identical locations in 55 testes of 29 men with non-obstructive azoospermia: (i) the rete testis region, ii) the midline, and (iii) the proximal region of the testis. When sperm cells were detected, they were used for intracytoplasmic sperm injection (ICSI), and the remainder were then cryopreserved in as many aliquots as possible (adjusted for ICSI procedure). Spermatozoa were found in 28 testes (50.9%) of 18 men (62.1%). In the testes from which spermatozoa were obtained, they were present in three, two or one locations in 15 (53.6%), five (17.9%) and eight (28.6%) cases respectively. The possibility of finding spermatozoa was not influenced by the location in the testis. Multiple testicular sperm extraction is recommended in cases of non-obstructive azoospermia, since it may enhance diagnostic accuracy of absolute testicular failure and increase the number of sperm cells retrieved.

Early ionic events in activation of the mammalian egg.

At fertilization of the mammalian egg, the spermatozoon initially binds to and then fuses with the egg plasma membrane. This critical event activates specific biochemical pathways within the egg. Activation of the egg induces resumption of meiosis and the start of rapid embryonic mitotic divisions on the one hand, and cortical granule exocytosis leading to modification of the zona pellucida and a block to polyspermy on the other. It has been shown in different systems that changes in intracellular ion concentrations can serve as second messengers of signal transduction mechanisms. The use of specific fluorescence probes, combined with the image analysis technique, facilitates the measurement of their dynamics in real time in the living cell and, thereby, assessment of their role in activation of the mammalian egg. This review focuses on the dynamics of intracellular Ca2+ and pH and their role in transducing the sperm signal to downstream cell cycle regulators.

High levels of anticardiolipin antibodies in patients with abnormal embryo morphology who attended an in vitro fertilization program.

PROBLEM: Recently, it has been suggested that anticardiolipin antibodies (ACAs) may serve as possible markers for reproductive failure. The association between ACAs and embryo morphology in patients undergoing in vitro fertilization (IVF) was investigated. METHOD OF STUDY: This prospective study comprised 117 patients with either tubal factor or unexplained infertility. Embryo morphology was blindly scored from I to IV according to blastomere regularity and the presence of fragments. Anticardiolipin antibodies (immunoglobulin [Ig] G and IgM) were detected. RESULTS: Anticardiolipin antibodies were found in 26 (50%) of the 52 patients with abnormal morphology, compared with 13 (20%) of the 65 patients with normal embryo morphology (P = 0.001). No statistically significant differences were found between the prevalence of ACAs among patients with tubal factor and those with unexplained infertility (29.6% and 36.5%, respectively). CONCLUSIONS: Our study shows an association between embryo morphology and the presence of ACAs. This association may explain the low implantation rate and early pregnancy loss in patients with ACAs.

Segregation of the pathways leading to cortical reaction and cell cycle activation in the rat egg.

At fertilization of the mammalian egg, resumption of the cell cycle and the cortical reaction are two events of egg activation, correlated with an increase in intracellular Ca2+ concentration and activation of protein kinase C. To evaluate the pathways leading to both events, rat eggs were parthenogenetically activated by the calcium ionophore ionomycin, or by the protein kinase C activators 12-O-tetradecanoyl phorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG). Cortical granule exudate was visualized by the lectin Lens culinaris and Texas Red streptavidin, using a confocal microscope. Resumption of meiosis was detected by Hoechst dye, and intracellular Ca2+ concentration by fura-2. Ionomycin triggered both a cortical reaction and resumption of meiosis, while chelation of intracellular Ca2+ rise by BAPTA-AM (1,2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester) revealed a segregation between these two events. A low Ca2+ transient (approximately 150 nM) induced a partial cortical reaction in half of the eggs, but the meiotic status was not affected. TPA triggered a cortical reaction with neither resumption of meiosis nor intracellular Ca2+ rise, while OAG induced both aspects of activation, as well as a significant intracellular Ca2+ rise. We conclude that in the cascade of events leading to egg activation, the initial Ca2+ rise is followed by a segregation in the pathway. A relatively low Ca2+ rise is sufficient to induce a partial cortical reaction. However, a higher level of Ca2+ is required to complete the cortical reaction and resumption of meiosis. The activation of the cell cycle is Ca2+-dependent, but protein kinase C-independent.

Tyrosyl-phosphorylated proteins are involved in regulation of meiosis in the rat egg.

Fertilization in invertebrates results in tyrosine (Tyr) phosphorylation of several egg proteins. However, the involvement of Tyr phosphorylation in mediating mammalian egg activation has not yet been investigated. Using an antibody specific for phosphotyrosine (P-Tyr), immunoblotting, and densitometric analysis, we found that maturation of the oocyte is accompanied by a generalized increase in the P-Tyr content of almost all egg proteins detected. After sperm penetration, 5 of the 17 protein bands detected demonstrated a small increase in their P-Tyr content, while at the pronuclear (PN) stage the signal was markedly reduced. Ionomycin emulated the changes observed at fertilization in most protein bands detected, demonstrating a small increase in their P-Tyr content within 15 min of exposure. Analysis of the involvement of the tyrosyl-phosphorylated, mitogen-activated protein (MAP) kinase during meiosis revealed comigration of the phosphotyrosyl bands with the protein and a good correlation with its enzyme activity. Maturation was accompanied by an increase in MAP kinase activity. The activity dropped partially after sperm penetration and furthermore later at the PN stage. A larger quantity accompanied by a more significant change in the P-Tyr content implies for extracellular regulated kinase (ERK) 2 being the dominant isoform present in the rat egg. Our results indicate that fertilization in mammals involves changes in activity of protein tyrosine kinases (PTKs) or in the balance between PTKs and protein tyrosine phosphatases. The single, ionomycin-induced Ca2+ rise is sufficient to imitate fertilization-induced changes in MAP kinase activity, as well as in tyrosine phosphorylation of other proteins within the egg.

Changes in calpain during meiosis in the rat egg.

Resumption of meiosis at fertilization is mediated by increased levels of calcium which activate several calcium-dependent enzymes. Calpain, a neutral calcium-activated thiol protease, is present in the cytoplasm of many cells. Its activation is associated with limited autolysis and relocalization in the cell. Calpain is thought to participate in the regulation of mitosis and resumption of meiosis in Xenopus oocytes. In this study we followed the activation and localization of calpain during maturation and fertilization in rat eggs using a polyclonal antibody raised against chicken muscle calpain. A band of 80 kDa was detected in GV oocytes and its level increased in unfertilized MII eggs. At the early stages of fertilization, we observed a transient decrease in the level of calpain which was regained at the pronuclear stage. Adding Ca2+ to lysate of MII eggs resulted in an additional band, representing the degraded fragment of the activated protein. In eggs activated by ionomycin, calpain level decreased, followed by an increase in a dynamic similar to that observed in fertilized eggs. Egg activation also led to changes in calpain localization. A homogenous distribution was observed in GV and in MII eggs, while in activated eggs it was localized predominantly overlying the metaphase plate. In the current study we demonstrate the presence of calpain in the rat egg. During maturation, calpain level increases; however, during egg activation, in response to [Ca2+]i changes, calpain undergoes autolysis, translocaton, and fluctuation in its level. We therefore suggest a correlation between calpain activation and fertilization.

Interleukin-10 in preovulatory follicular fluid of patients undergoing in-vitro fertilization and embryo transfer.

PROBLEM: The present study aimed at investigating the presence of Interleukin-10 (IL-10) in preovulatory follicular fluid (FF) and its possible correlation with 17-beta-estradiol (E2) and progesterone (P) levels, and treatment outcome in patients undergoing in-vitro fertilization-embryo transfer (IVF-ET). METHODS: Twenty consecutive patients with tubal factor infertility who underwent oocyte retrieval for IVF-ET were assayed for pooled, preovulatory FF levels of IL-10, E2, and P. RESULTS: The mean FF levels of IL-10, E2, and P were 78.7 +/- 104.7 pg/ml, 2,787.0 +/- 726.1 pg/ml, and 1.5 +/- 0.8 ng/ml, respectively. No correlation was found between preovulatory FF concentration of IL-10, E2, oocyte number, oocyte fertilization rate, embryo quality, and pregnancy rate. The levels of IL-10 were found to be negatively correlated with P concentration, although not significantly (P = 0.057). CONCLUSION: Interleukin-10 exists in the preovulatory FF. Further investigations are needed to determine the role of IL-10 in the folliculogenesis.

Intracellular pH of rat eggs is not affected by fertilization and the resulting calcium oscillations.

Penetration of the oocyte by a spermatozoon is the first in the series of events resulting in the transition of the egg from a quiescent to a proliferative state. A critical regulatory role for intracellular calcium ([Ca2+]i) ion activity has been demonstrated in all species studied so far. On the other hand, it has been demonstrated that the intracellular pH (pHi) changes, but only in a small number of species. This change also has been proposed as one of the most important events in egg activation. The present study was undertaken to monitor pHi in rat eggs during fertilization, using the membrane-permeable indicator BCECF-AM and fluorescence ratio imaging. Furthermore, we proposed to evaluate the relationship between pHi and [Ca2+]i changes during egg activation. We found that the ovulated rat egg has a cytoplasmic pH significantly different from that of the follicular oocyte. Insemination with capacitated sperm resulted in a microscopically visible sperm attachment, yet no change in pHi was observed. Eggs double-loaded with fura-2-AM and BCECF-AM before insemination were used to measure [Ca2+]i and pHi simultaneously. Eggs with a normal pattern of [Ca2+]i transients (i.e., fertilized eggs) did not show any change in pHi at least for 30 min following sperm binding. Data for eggs fertilized in vivo were recorded at later times after sperm binding; these served to exclude the possibility of a transient change that occurs between sperm-egg interaction and the pronuclear stage. We conclude that the pHi of rat eggs does not change during fertilization and therefore that fertilization-induced [Ca2+]i changes do not affect pHi in these eggs.

Low temperature and fertilization-induced Ca2+ changes in rat eggs.

In mammalian eggs, activation by sperm that leads to resumption of meiosis is characterized by an explosive transient increase in intracellular calcium ion concentration ([Ca2+]i), followed by [Ca2+]i oscillations. In addition to the spermatozoon, various treatments can induce parthenogenetic activation, accompanied by an elevation of [Ca2+]i. It has been reported that cooling can induce egg activation, yet the mechanism of this phenomenon has not been elucidated. In the present study we followed changes in egg [Ca2+]i (measured by Fura-2 fluorescence ratio imaging) during activation by cooling, using conditions that ensure a low rate of spontaneous activation. Our present findings demonstrate that cooling induces egg activation as manifested by [Ca2+]i transient(s) and second polar body extrusion. Seventy-eight of 104 eggs responded to cooling with increased [Ca2+]i. Thirty-five percent of the responding eggs displayed a single [Ca2+]i transient, while 65% exhibited at least two [Ca2+]i transients within the time window of the experiment (30-40 min). Twenty-two percent of these eggs displayed high-frequency oscillations (intervals of 3.5-5.9 min). In these eggs, the overall pattern of calcium dynamics was similar to that observed in eggs activated by sperm, as judged by the transient's intervals, duration, and a gradual increase in the amplitude of successive transients. The amplitudes of [Ca2+]i transients, however, were 2-3 times lower. We propose that cooling affects [Ca2+]i homeostasis to produce fertilization-like changes in [Ca2+]i, possibly associated with parthenogenetic activation. Moreover, great care should be exercised to prevent temperature changes during egg handling.

Restraint stress-induced changes in lymphocyte subsets and the expression of adhesion molecules.

The effect of restraint stress on the distribution of lymphocyte subsets were studied in young BALB/c male mice. Loss of whole body weight, a reduction in the weights of spleen and lymph nodes, and higher levels of serum corticosterone were evident after a single continuous restraint period of 16-18 h. Tissue sections of spleens from restrained animals revealed erythrocyte depletion in the contracted red pulp. Furthermore, there was a significantly higher proportion of CD4+, but not of CD8+, lymphocytes in the spleen. The proportion of the CD4+ subset was markedly diminished in peripheral blood, whereas no changes were detected in lymph nodes. Restraint resulted in enhanced allogeneic mixed lymphocyte reactivity and in altered expression of some CD4+, but not CD8+, splenocyte adhesion molecules (CD44, LFA-1 and VLA-4). Removal of circulating corticosteroids by surgical adrenalectomy abolished the restraint-induced changes in lymphocyte adhesion molecule expression. The findings suggest that the observed differences in lymphocytes subset distribution of lymphoid organs may be due to changes in the pattern of adhesion molecule expression.

Fertilization and early development of rat oocytes induced to mature by forskolin.

Forskolin has been shown to successfully induce maturation of rat oocytes as assessed by morphological markers. The present study was designed in an attempt to elucidate whether oocytes, induced to mature by forskolin (10(-4) M, group A) in a follicle-enclosed oocyte culture, are fertilizable and can further develop into two-cell embryos. Oocytes exposed in vitro to either luteinizing hormone (LH, 5 micrograms/ml, group B) or a GnRH agonist analogue (10(-7) M, group C) as well as oocytes that underwent maturation in vivo (group D), served as positive controls. We found that similar rates of fertilization were obtained in the experimental and all of the above mentioned control groups (A = 78.9 +/- 4.2%, B = 77.9 +/- 3.1%, C = 77.5 +/- 5.5% and D = 84.7 +/- 2.7%). Cleavage rate of fertilized eggs from group A was significantly higher than that of eggs from groups B & C, and similar to that of eggs from group D (A = 63.1 +/- 6.7%, B = 37.8 +/- 4.9%, C = 50.0 +/- 4.1%, D = 67.8 +/- 4.1%). Using functional parameters we hereby demonstrate that forskolin and LH are at least equally potent in producing fertilizable eggs that have a high potential of development into two cell embryos. These results further support the idea that cAMP is a mediator of LH action in inducing oocyte maturation.