Effective repair of articular cartilage using human pluripotent stem cell-derived tissue.

In an effort to develop an effective source of clinically relevant cells and tissues for cartilage repair a directed differentiation method was used to generate articular chondrocytes and cartilage tissues from human embryonic stem cells (hESCs). It has previously been demonstrated that chondrocytes derived from hESCs retain a stable cartilage-forming phenotype following subcutaneous implantation in mice. In this report, the potential of hESC-derived articular-like cartilage to repair osteochondral defects created in the rat trochlea was evaluated. Articular cartilage-like tissues were generated from hESCs and implanted into the defects. After 6 and 12 weeks, the defects were evaluated histologically and immunohistochemically, and the quality of repair was assessed using a modified ICRS II scoring system. Following 6 and 12 weeks after implantation, hESC-derived cartilage tissues maintained their proteoglycan and type II collagen-rich matrix and scored significantly higher than control defects, which had been filled with fibrin glue alone. Implants were found to be well integrated with native host tissue at the basal and lateral surfaces, although implanted human cells and host cells remained regionally separated. A subset of implants underwent a process of remodeling similar to endochondral ossification, suggesting the potential for a single cartilaginous implant to promote the generation of new subchondral bone in addition to repair of the articular cartilage. The ability to create cartilage tissues with integrative and reparative properties from an unlimited and robust cell source represents a significant advance for cartilage repair that can be further developed in large animal models before clinical- setting application.

EGF/ErbB receptor family in ovarian cancer.

In summary, the EGF/ErbB family of receptor tyrosine kinases has been shown to play a key role in normal ovarian follicle development, and cell growth regulation of the ovarian surface epithelium. Disregulation of these normal growth regulatory pathways, including overexpression and/or mutation of EGFR/ErbB receptor family members, as well as elements of their downstream signalling pathways, have been shown to contribute to the etiology and progression of epithelial ovarian cancer. It is, therefore, not surprising that these gene products, and their related soluble receptor isoforms may have clinical utility as tumor and/or serum biomarkers of disease activity. Moreover, since several of these soluble receptor isoforms have potent growth inhibitory activity, and are naturally occurring in the circulation, they are ideal candidates for the development of novel therapeutics for the treatment of ovarian cancer patients.

Lack of acrosome formation in Hrb-deficient mice.

The sperm acrosome is essential for sperm-egg fusion and is often defective in men with nonobstructive infertility. Here we report that male mice with a null mutation in Hrb are infertile and display round-headed spermatozoa that lack an acrosome. In wild-type spermatids, Hrb is associated with the cytosolic surface of proacrosomic transport vesicles that fuse to create a single large acrosomic vesicle at step 3 of spermiogenesis. Although proacrosomic vesicles form in spermatids that lack Hrb, the vesicles are unable to fuse, blocking acrosome development at step 2. We conclude that Hrb is required for docking and/or fusion of proacrosomic vesicles during acrosome biogenesis.

Defects in the germ line and gonads of mice lacking connexin43.

The connexins are a family of at least 15 proteins that form the intercellular membrane channels of gap junctions. Numerous connexins, including connexin43 (Cx43), have been implicated in reproductive processes by virtue of their expression in adult gonads. In the present study, we examined the gonads of fetal and neonatal mice homozygous for a null mutation in the Gja1 gene encoding Cx43 to determine whether the absence of this connexin has any consequences for gonadal development. We found that in both sexes at the time of birth, the gonads of homozygous mutants were unusually small. This appears to be caused, at least in part, by a deficiency of germ cells. The germ cell deficiency was traced back as far as Day 11.5 of gestation, implying that it arises during early stages of germ line development. We also used an organ culture technique to examine postnatal folliculogenesis in the mutant ovaries, an approach necessitated by the fact that Gja1 null mutant offspring die soon after birth because of a heart abnormality. The results demonstrated that folliculogenesis can proceed to the primary (unilaminar) follicle stage in the absence of Cx43 but that subsequent development is impaired. In neonatal ovaries of normal mice, Cx43 could be detected in the somatic cells as early as Day 1, when primordial follicles begin to appear, supporting the conclusion that this connexin is required for the earliest stages of folliculogenesis. These results imply that gap junctional coupling mediated by Cx43 channels plays indispensable roles in both germ line development and postnatal folliculogenesis.

Altered gap junctional communication, intercellular signaling, and growth in cultured astrocytes deficient in connexin43.

Astrocytes are characterized by extensive intercellular communication mediated primarily by gap junction channels composed of connexin43. To examine this junctional protein in astrocytic functions, astrocytes were cultured from embryonic mice with a null mutation in the connexin43 gene (Reaume et al.: Science 267:1831-1834, 1995). Using anti-Cx43 antibodies, immunoblotting and immunostaining indicated that homozygous null astrocytes were devoid of Cx43. They are also deficient in intercellular dye transfer. Astrocytes cultured from heterozygous embryos express significantly lower Cx43 compared to wild type, and their dye coupling is reduced. Markers of glial differentiation, such as glial fibrillary acidic protein and S100, appeared similar in all genotypes. Measurement of intercellular calcium concentration following mechanical stimulation of confluent astrocytes revealed that the number of cells affected by a rise in intracellular calcium was reduced in homozygous cultures compared to wild type. In fact, the calcium response in homozygous astrocytes was similar to that observed in wild-type astrocytes in the presence of a gap junction blocker. The growth rate of astrocytes lacking Cx43 was reduced compared to wild-type astrocytes. These results suggest that gap junctional intercellular communication mediated by Cx43 is not critical for astrocyte differentiation but is likely involved in the regulation of intercellular calcium signaling and cell growth.

Normal development of preimplantation mouse embryos deficient in gap junctional coupling.

The connexin multigene family (13 characterized members in rodents) encodes the subunits of gap junction channels. Gap junctional intercellular coupling, established during compaction of the preimplantation mouse embryo, is assumed to be necessary for development of the blastocyst. One member of the connexin family, connexin43, has been shown to contribute to the gap junctions that form during compaction, yet embryos homozygous for a connexin43 null mutation develop normally, at least until implantation. We show that this can be explained by contributions from one or more additional connexin genes that are normally expressed along with connexin43 in preimplantation development. Immunogold electron microscopy confirmed that roughly 30% of gap junctions in compacted morulae contain little or no connexin43 and therefore are likely to be composed of another connexin(s). Confocal immunofluorescence microscopy was then used to demonstrate that connexin45 is also assembled into membrane plaques, beginning at the time of compaction. Correspondingly, embryos homozygous for the connexin43 null mutation were found to retain the capacity for cell-to-cell transfer of fluorescent dye (dye coupling), but at a severely reduced level and with altered permeability characteristics. Whereas mutant morulae showed no evidence of dye coupling when tested with 6-carboxyfluorescein, dye coupling could be demonstrated using 2',7'-dichlorofluorescein, revealing permeability characteristics previously established for connexin45 channels. We conclude that preimplantation development in the mouse can proceed normally even though both the extent and nature of gap junctional coupling have been perturbed. Despite the distinctive properties of connexin43 channels, their role in preimplantation development can be fulfilled by one or more other types of gap junction channels.

Postsurgical intraperitoneal exposure to glove powders modulates inflammatory and immune-related cytokine production.

The objective of the present study was to determine whether intraperitoneal exposure to glove powders modulates the inflammatory and immune responses by altering the influx of inflammatory and immune cells and peritoneal fluid cytokines and thus the outcome of surgically induced peritoneal wound healing. Peritoneal wall injuries were made by scraping the tissue until bleeding occurred in 360 mice. One of the following fluids was then introduced into the peritoneal cavity: phosphate-buffered saline solution, phosphate-buffered saline solution containing glove powders (Biosorb and Keoflo, 100 microg/ml), Hydrocote (Hydrogel film, Biogel 100 microg/ml), latex proteins (1 mg/ml), or lipopolysaccharides (12.5 microg/ml). At intervals of 1 to 28 days after injury, 10 mice per treatment per day and 10 uninjured mice were killed, peritoneal fluids were collected to determine the cytokine levels, the rate of fibrous adhesions formed at the site of injuries was graded, and peritoneal walls with attached fibrous adhesions were removed to determine the degree of inflammatory and immune cell infiltration into the wound. The results indicated that, with the exception of interferon-gamma, the peritoneal fluid levels of transforming growth factor-beta1, tumor necrosis factor-alpha, interleukin-1beta, and granulocyte-macrophage-colony stimulating factor in the phosphate-buffered saline solution-treated injured group significantly increased, reaching maximum between days 4 and 7 (p < 0.05) compared with the uninjured group and returned to uninjured values by day 14 after injury. The level of transforming growth factor-beta1 was higher in glove powders and Hydrocote-treated groups than in latex, lipo-saccharides, or phosphate-buffered saline solution-treated groups until day 14 after surgery (p < 0.05). The levels of tumor necrosis factor-alpha and interleukin-1beta increased in all treatment groups during the first week after injury compared with uninjured controls, with the exception of Hydrocote. The number of T helper/inducers (CD4), total leukocytes (CD11a), B lymphocytes (CD45R), granulocytes (Gr-1), and mononuclear phagocytes (Mac-3) in the wound increased during the first week after peritoneal wounding with no significant difference between treated and untreated groups. The rate of adhesion formation was not significantly altered in treated compared with untreated groups. These data suggest that a mechanism which mediates glove powder-induced peritoneal inflammatory and immune reactions in the postsurgical setting involves augmentation of cytokine production without influencing the influx of inflammatory and immune cells or adhesion formation.

Ovarian intrabursal administration of transforming growth factor beta 1 inhibits follicle rupture in gonadotropin-primed mice.

We hypothesized that over-expression and/or activation of latent transforming growth factor betas (TGF betas) by various ovarian cell types may lead to disturbances in ovulation and fertilization. To test this hypothesis, active TGF beta ranging from 1 to 500 ng was administered intrabursally into the ovaries of gonadotropin-primed mice, and the rates of ovum recovery and fertilization were determined. Furthermore, the presence and cellular distribution of endogenous TGF betas and TGF beta type I and type II receptors were determined immunohistochemically in the ovarian tissues of TGF beta 1-treated and untreated groups. The total number of ova recovered per ovary from ovaries treated as pairs or treated singly with TGF beta 1 at 1 to 10 ng/ovary was similar to that from controls, whereas the number recovered from ovaries treated as pairs or singly with 50 or 100 ng of TGF beta 1 per ovary was significantly lower than the number from respective controls (p < 0.05, 0.001). The number of ova recovered per ovary from ovaries treated as pairs or singly with TGF beta 1 at 200 or 500 ng/ovary was similar to the number of ova obtained from ovaries treated with TGF beta 1 at 100 ng/ovary. The rate of in vitro fertilization was low in ova recovered from ovaries treated with 50, 100, 200, and 500 ng/ovary of TGF beta 1, compared to that in ova from untreated ovaries. Histologically, the TGF beta 1-treated ovaries contained large numbers of unruptured follicles, whereas untreated ovaries contained large numbers of corpora lutea. Immunohistochemically, the endogenous TGF beta 1 and TGF beta 2 was localized in theca, granulosa, and luteal cells, without a substantial difference in intensity or distribution, in both TGF beta 1-treated ovaries and in controls. Theca cells were the primary site of immunoreactive TGF beta protein. TGF beta type I and type II receptors were also present in these cells, and their relative immunoreactive intensity was considerably reduced, particularly in granulosa cells in TGF beta 1-treated ovaries compared to controls. The results support our hypothesis and suggest that TGF betas play an important regulatory role in follicular development, oocyte maturation, and the ovulatory process.

Development of astrocytes and neurons in cultured brain slices from mice lacking connexin43.

Astrocyte and neuronal development was investigated in organotypic brain slice cultures from mouse fetuses with a null mutation in the connexin43 gene. Astrocyte morphology and electrical properties were indistinguishable in null mutant slices and control slices but at 18 days in vitro astrocyte density in the central regions of the null mutant slices was significantly higher than in control slices. Neuronal development assessed morphologically and electrophysiologically appeared normal in the mutant slices. These results suggest that intercellular communication mediated through connexin43 is not essential for the development of astrocytes and neurons but may play a role in regulating astrocytic migration.

Contraception potential of neem oil: effect on pregnancy success in the mouse.

PURPOSE: The aim of this study was to find out the role and mechanism of action of neem oil as a postcoital fertility blocker in mouse. METHODS: Female mice were injected with neem oil (20 or 40 microliters) surgically into each uterine horn on day 2 postcoitum (pc). Both the uterine horns of each mouse were injected. Arachis oil served as vehicle control. Pregnancy success was determined by the number of implanted embryos on day 8 pc and the number of live fetuses in the uteri on day 18 pc. Transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), and epidermal growth factor receptor (EGFR) were immunolocalized in the paraffin-embedded sections of the uteri at 0600 hr on day 5 pc. The unimplanted embryos were assessed in the uteri at 2000 hr on day 5 pc. Uterine secretions were assessed for the leukocytes infiltration on day 4 through day 8 pc. RESULTS: The number of implantation sites on day 8 pc and the number of live fetuses on day 18 pc were lower in the neem oil-treated animals compared to their respective control animals at both the concentrations of neem oil (20 and 40 microliters/uterine horn). Neem oil also caused resorption of some embryos between day 8 pc and day 18 pc. In neem oil-treated mice, EGFR immunostaining decreased in the luminal and glandular epithelium and increased in the stroma as determined at 0600 hr on day 5 pc. Uterine secretions on day 4 through day 6 pc from the neem oil-treated mice showed massive leukocyte infiltration. Unimplanted preimplantation embryos, underdeveloped, degenerated, or at blastocyst stage, were recovered from the uteri after flushing at 2000 hr on day 5 pc from the neem oil-treated animals. A number of retrieved unimplanted embryos showed the direct attachment of the leukocytes to their zona pellucida. It is believed that the secretions of these leukocytes might be responsible for the underdevelopment of the early embryos and hence inhibition of implantation. The exact interaction of these leukocytes and their secretions with the early embryos is under investigation. CONCLUSIONS: Postcoital intrauterine treatment of neem oil during preimplantation period causes fertility block in mouse by lowering the EGFR localization in the luminal and glandular epithelium, by causing massive leukocytes infiltration into the uteri, by degenerating the early embryos, and by causing the postimplantation embryonic resorptions in the uteri. The possible mechanism of action of neem oil is discussed.

Effect of anordrin on the development of mouse preimplantation embryos in vitro.

PURPOSE: The in vitro effect of anordrin and anordiol on the development of mouse two-cell embryos was studied. METHOD: Female mice were primed with gonadotropins for superovulation and caged with male mice. Preimplantation embryos, at the two-cell stage, were recovered from the oviducts at 40 hr post-hCG. In the first experiment, two-cell embryos were exposed to culture medium containing different concentrations of anordrin for 3, 12, 24, and 80 hr and then grown in the anordrin-free culture medium and assessed for the formation of total and hatching blastocysts at 80 hr. In the second experiment, two-cell embryos were grown in culture medium containing different concentrations of anordiol and assessed for the formation of total and hatching blastocysts at 80 hr in vitro. RESULTS: Exposure of two-cell embryos to anordrin concentrations of 2.5-7.5 micrograms/ml for 12 hr, 2.5-5.0 micrograms/ml for 24 hr, and 2.5 micrograms/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 2.5-7.5 micrograms/ml for 12 hr, 1.0-2.5 micrograms/ml for 24 hr, and 1.0 micrograms/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts, in a exposure time-dependent and dose-dependent manner. Exposure of two-cell embryos to anordiol concentrations of 15-25 micrograms/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 15-20 micrograms/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts in a dose-dependent manner. CONCLUSION: Anordrin and its metabolite anordiol inhibit the development of two-cell embryos in vitro.

Influence of hyperthyroidism induced at prepuberty on the epididymal lipids, number and motility of spermatozoa in rats.

Fifteen prepubertal rats were divided into 3 groups of 5 each: (1) hyperthyroid group (TH), the rats were treated with thyroxine (T4) (25 micrograms/100g body weight/day) for 60 days from the age of day 31 to day 90; (2) T4-withdrawal group (TH-T4), the rats were given T4 for 30 days from the age of day 31 to day 60 and then the T4 treatment was withdrawn for the following 30 days from the age of day 61 to day 90 and the animals were given vehicle during this period; and (3) control group, the rats were given vehicle for 60 days from the age of day 31 to day 90. All were killed at the age of day 91. Serum levels of testosterone, T4 and triiodothyronine increased in the TH group (P < 0.001). The levels decreased to the euthyroid status in the TH-T4 group. Hyperthyroidism caused various changes in the levels of epididymal lipids. The levels changed further or were restored differentially in the TH-T4 group. The number of spermatozoa decreased, in caput epididymis (CpE) as well as in cauda epididymis (CdE), in the TH group (P < 0.001). The number was not restored in the TH-T4 group. The forward motility of the spermatozoa, determined in CdE, decreased in the TH group (P < 0.001) and was not restored in the TH-T4 group. The study showed that chronic hyperthyroidism, induced at the prepubertal age, causes various changes in epididymal lipid composition and sperm parameters. The study may be helpful in revealing the reasons for male infertility in hyperthyroid patients.

Cardiac malformation in neonatal mice lacking connexin43.

Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43 (Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart. This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among connexins in other parts of the developing fetus.

Ova recovery, and in vivo and in vitro fertilization of ova from RU486-treated PMSG/hCG-primed mice.

In this study, the effect of preovulatory treatment with RU486, for different lengths of time and combinations of days, was shown in terms of the ova recovery, in vitro fertilization of recovered ova, in vivo fertilization and quality of fertilized ova in PMSG/hCG-primed mice. Female mice were injected with PMSG followed 48 h later by hCG to induce superovulation. Mice received RU486 (20 mg kg-1 body wt) for 1, 2, 3 and 4 preovulatory days (in different combinations). Ovulation, as judged by the number of ova recovered at 14 to 14.5 h post-hCG, was depressed (P < 0.001), and the total number of embryos recovered at 40 h post-hCG was low (P < 0.001), in mice receiving a minimum of two consecutive days' treatment (day before PMSG + day of PMSG; or day before hCG + day of hCG) of RU486 under study. Quality of ova recovered from RU486-treated animals was not affected as determined by their ability to become fertilized in vitro. In vivo fertilization, as determined by the recovery of 2-cell embryos, was suppressed significantly in mice treated with RU486 for four consecutive preovulatory days (P < 0.001). A varied degree of premature compaction was observed in 2-cell embryos immediately upon retrieval from the oviduct of RU486-treated animals, the effect being most marked in mice receiving RU486 for a minimum of two consecutive preovulatory days under study. It is suggested that premature compaction of early embryos was under the continuous influence of the luminal environment of treated animals and might be the reason for their degeneration at later stages in the reproductive tract and for a low pregnancy rate as shown by other studies. Compacted embryos decompacted within 15-30 min in vitro and led to normal blastocyst formation in vitro in RU486-free culture medium.

Modulation of mouse sperm-egg interaction, early embryonic development and trophoblastic outgrowth by activated and unactivated macrophages.

Exposure of mouse spermatozoa and oocytes duringin vitro fertilization (IVF) to lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) activated macrophages (U937 cell line), but not unactivated macrophages cultureconditioned medium or control medium (RMPI+DMEM with 0.5% FBS) resulted in inhibition of IVF (87.2%), first cleavage (90.8%) and total blastocyst formation 97.5%). The direct coculture of the activated macrophages with 2-cell stage embryos resulted in arrested development (91.2%), an effect that was significantly diminished in the presence of monolayer of human endometrial stromal cells in the coculture (58.3%). In contrast, the majority of 2-cell embryos developed to blastocysts when exposed to unactivated macrophages, or macrophage-stromal cell cocultures (94.1%). The majority of 2-cell embryos cultured in control medium (DMEM/Ham's F12 with 2% FBS) developed to morulae (96.2%), then underwent growth arrest and degeneration. Furthermore, culturing blastocyst stage embryos in the above groups resulted in a significant enhancement of trophoblast outgrowth, particularly in coculture with activated macrophages as compared to any other group (P<0.005). There was a significant increase in the levels of TGF-ß, GM-CSF, IL-1α, IL-1ß, TNF-α, PGE(2), TXB(2) and LTB(4) released into the culture conditioned medium of activated macrophages compared to unactivated macrophages (P<0.001). These results suggest that the secretory products of activated macrophages, among them those determined in this study, in a stage-specific manner can directly effect sperm-egg interaction, early embryonic development and trophoblastic outgrowth. This data provides further support for the hypothesis that in endometriosis-associated infertility, continuous exposure of spermatozoa, oocytes and early embryos to activated macrophage-derived factors may play a vital role in their survival during transportation and fertilization as well as development during early embryonic stage.

Influence of hypothyroidism induced at prepuberty on epididymal lipids and the number and motility of spermatozoa in rats.

Fifteen prepubertal male rats (age 30 days) were divided into three groups of five each: Group 1, hypothyroid (Tx)--rats were thyroidectomized at day 30; Group 2, T4 (L-thyroxine) replacement therapy (Tx+T4)--rats were thyroidectomized at day 30 and treated daily i.m. with T4 (6 micrograms/100 g body weight/day) for 30 days from day 31 to day 60 post-thyroidectomy (age 90 days); Group 3, control--rats were sham-operated and treated with vehicle. The rats from all groups were killed on day 61 post-thyroidectomy or post-sham operation (age 91 days). The serum levels of testosterone, T4 and T3 decreased in the Tx group (p < 0.001). In the Tx+T4 group the levels of T4 and T3 were restored to control values, whereas testosterone levels remained lower than in the control group. Hypothyroidism caused various changes in the levels of epididymal phospholipids and neutral lipids. These were restored differentially or were altered further in the Tx+T4 group. The number and forward motility of spermatozoa, recovered from the cauda epididymis, were decreased significantly (p < 0.01) in the Tx group and were not restored in the Tx+T4 group. This study shows that chronic hypothyroidism, induced at prepuberty and continued for 60 days, causes various changes in the lipid composition of the caput and cauda epididymis and lowers the quality and quantity of spermatozoa in the cauda epididymis. T4 therapy for 30 days, especially during the postpubertal period, did not restore the quality or quantity of spermatozoa and caused differential changes in the levels of epididymal lipids, depending upon the region.(ABSTRACT TRUNCATED AT 250 WORDS)

Neem oil inhibits two-cell embryo development and trophectoderm attachment and proliferation in vitro.

PURPOSE: The in vitro effect of neem oil was studied on the development of mouse two-cell embryos and trophectodermal cell attachment and proliferation. METHOD: Female mice were primed with gonadotropins for superovulation and caged with male mice. Early embryos, at the two-cell and the blastocyst stages, were recovered at 40 and 88 hr post-hCG from the oviducts and the uteri, respectively. In the first experiment, two-cell embryos were exposed to culture medium containing different concentrations of neem oil for 1, 12, and 24 hr and then grown in neem oil-free culture medium and assessed for the formation of total and hatching blastocysts at 96 hr. In the second experiment, partially hatching blastocysts were cocultured with human endometrial stromal cell monolayers in culture medium containing different concentrations of neem oil and assessed for the attachment and proliferation of trophectodermal cells at 96 hr. RESULTS: Exposure of two-cell embryos to neem oil concentrations of 0.050-0.500% for 1 hr, 0.010-0.250% for 12 hr, and 0.005-0.100% for 24 hr caused significant inhibition of the formation of total and hatching blastocysts, in a dose-dependent manner. Neem oil at 0.050-0.100% concentrations inhibited, in a dose-dependent manner, the in vitro attachment and proliferation of trophectodermal cells of partially hatching blastocysts cocultured with human endometrial stromal cells monolayers. CONCLUSION: Neem oil inhibits the development of two-cell embryos and attachment and proliferation of the trophectodermal cells of partially hatching blastocysts in vitro. The study encourages the use of this herbal product as a postcoital contraceptive that warrants further research.

Successful pregnancy with reduced ovarian mass in the rat.

This paper reports the successful pregnancy in rats with only 10-20% of ovarian tissue. Sprague-Dawley female rats of breeding age were divided into five groups: group 1 (completely ovariectomized), the ovaries from both the sides of the rats were removed completely; group 2 (partially ovariectomized, 80-90% of the ovary of each side was removed leaving the remaining tissue in place; group 3 (ovariectomized with flank transplant), the ovary of each side was removed from the ovarian stalk and inserted into a subcutaneous pocket made surgically in the flank on the respective side of the same rat; group 4 (partially ovariectomized with flank transplants), 80-90% of the ovarian mass was removed from the ovarian stalk and put back in the flank position in the subcutaneous pocket on the same side; group 5 (control), rats with intact ovaries. Estrous cyclicity, mating behavior and pregnancy rate were recorded in the animals. Percent rats cycled were 0.00, 80.00, 80.00, 70.00 and 100 in groups 1-5 respectively; percent cycled rats mated were 86.27, 100, 71.42 and 100 in groups 2-5 respectively; and percent mated rats reaching successful pregnancy were 36.36, 0.00, 100 and 100 in groups 2-5 respectively. Pregnancy rate in the mated rats in group 2 was lower than that of groups 4 (P < 0.01) and 5 (P < 0.01), whereas, it did not differ among groups 4 and 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse sperm-egg interaction in vitro in the presence of neem oil.

In vitro evidence is presented showing toxicity of neem oil on sperm-egg interaction in mouse. Cumulus oophorus-enclosed ova, inseminated with capacitated spermatozoa, were cultured in 1 ml of in vitro fertilization (IVF) medium and overlayered by 1 ml of different concentrations of neem oil (1, 5, 10, 25, 50 and 100%) for IVF duration of 4h. At the end of incubation, ova were allowed to grow in neem oil-free culture medium and assessed for fertilization, first cleavage (2-cell formation) and blastocyst formation in vitro at 4-14h, 24h and 108h post-insemination respectively. The study showed that the presence of neem oil at concentrations of 10, 25 and 50% caused inhibition of IVF in a dose-dependent manner. The toxic effect of exposure of 25 and 50% neem oil was further carried over to the first cleavage of the resulting fertilized ova and the toxic effect of 5, 10, 25 and 50% was carried over to the blastocyst formation from the resulting fertilized ova when grown in neem-oil free culture medium. A total of 94.1% inhibition of 2-cell formation and 100% inhibition of blastocyst formation from the inseminated ova was observed in 50 and 25% neem oil-treated groups respectively. Neem oil at 100% concentration caused 100% degeneration of ova at 1h of sperm-ova coculture. The study showed a direct toxic effect of neem oil on sperm-egg interaction in vitro and encourages research investigations of this herbal product as a pre-coital contraceptive.

Effect of RU486 on different stages of mouse preimplantation embryos in vitro.

17 beta-Hydroxy-11 beta(4-dimethylaminophenyl)-17 alpha-(1-propynyl)estra-4, 9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 degrees C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 micrograms/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 micrograms/mL culture medium (p less than 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 microgram/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 micrograms/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.