Richard G. Rawlins

Modulation of ovarian follicle maturation and effects on apoptotic cell death in holtzman rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) in utero and lactationally

Recent reports have described the reproduction-modulating and endocrine-disrupting effects following exposure to toxic substances such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Herein, we set out (1) to determine whether TCDD exposure exerts detrimental effects on follicle maturation in the Holtzman rat ovary and (2) to determine whether the effects of TCDD are mediated in part via apoptotic cell death. In certain species, dioxin exposure is correlated with reduced fecundity, reduced ovulatory rate, an increased incidence of endometriosis, and various reproductive cancers. Although some of the effects of TCDD are mediated via the hypothalamic-pituitary axis, direct effects on the ovary have also been observed. In the present study, an oral dose of 1 microgram TCDD/kg maternal body weight was administered on Day 15 of gestation. Female pups were sacrificed on Postnatal Day 21/22, and the ovaries were excised, fixed for histologic analysis, and analyzed in a double-blind paradigm. The analysis included a count and measurement and classification of preantral and antral follicles throughout the entire ovary. The contralateral ovary from each animal was analyzed for DNA fragmentation indicative of apoptotic cell death. The results indicate that TCDD treatment significantly reduced the number of antral follicles in the size classes 50,000 to 74,999 microns2 and > 100,000 microns2. We also observed a reduction in the number of preantral follicles less than 50,000 microns2. No difference was observed in the degree of apoptotic cell death in antral (50,000 to > 100,000 microns2) and preantral follicles (50,000 microns2 to > 75,000 microns2) between TCDD-treated and control-treated tissues. These data support the hypothesis that TCDD results in a diminution in the number of antral and preantral follicles of certain size classes in animals exposed during critical periods of development, but that apoptosis does not appear to be the underlying mechanism in these particular follicles. This does not preclude apoptosis occurring in pools of smaller precursor follicles.

Identification of a novel gene set in human cumulus cells predictive of an oocyte's pregnancy potential

OBJECTIVE To identify a gene expression signature in human cumulus cells (CCs) predictive of pregnancy outcome across multiple clinics, taking into account the clinic and patient variations inherent in IVF practice. DESIGN Retrospective analysis of single human cumulus-oocyte complexes with the use of a combined microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) approach. SETTING Multiple private IVF clinics. PATIENT(S) Fifty-eight patients. Samples from 55 patients underwent qRT-PCR analysis, and samples from 27 patients resulted in live birth. INTERVENTION(S) Gene expression analysis for correlation with pregnancy outcome on individual human CCs collected immediately after oocyte retrieval. Pregnancy prediction analysis used leave-one-out cross-validation with weighted voting. MAIN OUTCOME MEASURE(S) Combinatorial expression of 12 genes in 101 samples from 58 patients. RESULT(S) We found a set of 12 genes predictive of pregnancy outcome based on their expression levels in CCs. This pregnancy prediction model had an accuracy of 78%, a sensitivity of 72%, a specificity of 84%, a positive predictive value of 81%, and a negative predictive value of 76%. Receiver operating characteristic analysis found an area under the curve of 0.763 ± 0.079, significantly greater than 0.5 (random chance prediction). CONCLUSION(S) Gene expression analysis in human CCs should be considered in identifying oocytes with a high potential to lead to pregnancy in IVF-ET.

Induction of the human sperm acrosome reaction by human oocytes

The acrosome reaction of human spermatozoa incubated in the presence or absence of vested human oocytes was investigated. All gametes were obtained from human in vitro fertilization (IVF) cases. Spermatozoa were collected after incubation in insemination medium only and following removal of the oocytes from insemination medium during the IVF procedure. After 16 hours of incubation 18.5% of the spermatozoa in insemination medium alone were acrosome-reacted compared to 31.5% for spermatozoa incubated in medium containing oocytes. The acrosome reaction of spermatozoa incubated with fertilized or unfertilized oocytes was also investigated. The percentage of acrosome reaction did not differ ( P > 0.05) between the two groups (29.7% in the fertilized cases versus 30.7% in the unfertilized cases). Completion of oocyte nuclear maturation did not affect the proportion of acrosome-reacted spermatozoa observed with unfertilized eggs. A similar ( P > 0.05) percentage of acrosome reacted spermatozoa were observed regardless of whether the unfertilized oocytes had (29%) or had not (35%) reached metaphase II. These findings indicate the acrosome reaction of human spermatozoa is enhanced in the presence of vested human oocytes. Furthermore, there is no apparent correlation between the percentage of the population of spermatozoa that acrosome react in the medium and the potential of an oocyte for fertilization.

Estrogen receptor and aromatic hydrocarbon receptor in the primate ovary.

We have previously shown by immunocytochemistry and autoradiography the presence of estrogen receptors (ER) in rhesus monkey ovary. Intense chromogen staining showed specific binding for ER in nuclei of germinal epithelium and granulosa cells of antral follicles; and radiolabeled ligand bound specifically to functional corpora lutea (CL). Although it is accepted that the germinal epithelium of the primate ovary contains ER, some controversy still persists regarding the intraovarian localization of this molecule. In addition, no data exist that localize the aromatic hydrocarbon (dioxin) receptor (AHR), which is known to modulate ER, to the primate ovary. In the present study, we show the presence of ER using Western blot analysis, and ER capable of binding DNA within intraovarian compartments in two species of the genusMacaca (rhesus macaque,Macaca mulatta and stumptail macaque,Macaca arctoides); extend these findings to human ovarian granulosa cells (GC) using Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and gel mobility-shift analysis; and localize the AHR to intraovarian compartments of the macaque ovary by Western blots and gel-shift assays. These experiments strongly suggest that estrogens can exert effects on follicle development directly at the ovary, and provide the first direct evidence that AHR-mediated toxicity may be manifested at the ovary to induce possible antifertility effects.

First pregnancies and livebirths from transfer of sodium alginate encapsulated embryos in a rodent model

OBJECTIVE To determine the effect of sodium alginate encapsulation of rodent embryos on in vitro embryonic cleavage rates, implantation rates, and livebirth rates, and to find the in vivo degradation time for the capsules. DESIGN Studies were conducted using both CB6F1 mice and Golden Syrian hamsters. RESULTS Capsules made with 3.0% sodium alginate degraded in vivo within 24 to 48 hours after transfer. In vitro embryonic cleavage of encapsulated embryos was not impaired, nor were implantation rates in CB6F1 mice. Finally, 8.6% of transferred encapsulated embryos resulted in livebirths. CONCLUSIONS Encapsulation of rodent embryos in 3.0% sodium alginate is not detrimental to embryonic development, implantation rates, or fetal development. Because the capsule degrades within 48 hours after transfer, encapsulating embryos may be beneficial for human in vitro fertilization and embryo transfer.

Demographic variability and density-dependent dynamics of a free-ranging rhesus macaque population

Density‐dependence is hypothesized as the major mechanism of population regulation. However, the lack of long‐term demographic data has hampered the use of density‐dependent models in nonhuman primates. In this study, we make use of the long‐term demographic data from Cayo Santiagos rhesus macaques to parameterize and analyze both a density‐independent and a density‐dependent population matrix model, and compare their projections with the observed population changes. We also employ a retrospective analysis to determine how variance in vital rates, and covariance among them, contributed to the observed variation in long‐term fitness across different levels of population density. The population exhibited negative density‐dependence in fertility and the model incorporating this relationship accounted for 98% of the observed population dynamics. Variation in survival and fertility of sexually active individuals contributed the most to the variation in long‐term fitness, while vital rates displaying high temporal variability exhibited lower sensitivities. Our findings are novel in describing density‐dependent dynamics in a provisioned primate population, and in suggesting that selection is acting to lower the variance in the population growth rate by minimizing the variation in adult survival at high density. Because density‐dependent mechanisms may become stronger in wild primate populations due to increasing habitat loss and food scarcity, our study demonstrates that it is important to incorporate variation in population size, as well as demographic variability into population viability analyses for a better understanding of the mechanisms regulating the growth of primate populations. Am. J. Primatol. 75:1152–1164, 2013.

Effect of sodium alginate encapsulation on the development of preimplantation mouse embryos

Submitted: December 23, 1986 Accepted: August 4, 1987 (North American Editorial Office) plantation by protecting the zygote, to minimize the associated risk of ectopic pregnancy following embryo transfer by preventing tubal reflux, and to ensure correct placement of IVF embryos in utero. These objectives are met by encapsulating embryos in biodegradable sodium alginate prior to transfer. Previous work has shown that differentiated pancreatic cell lines remain viable in sodium alginate (6), but the method has not been applied to preimplantation embryos. The effects of encapsulation in sodium alginate were tested on two-cell mouse embryos, cultured with controls for 72 hr in vitro.

Effects of dioxin, an environmental pollutant, on mouse blastocyst development and apoptosis

OBJECTIVE To evaluate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) on mouse embryo development and apoptosis. DESIGN Controlled animal study. SETTING Academic research environment. ANIMAL(S) Female mice (CB6F1) at 3 to 6 weeks of age and proven breeders (C578B46). INTERVENTION(S) Mouse embryos were obtained at the morula stage and cultured to the blastocyst stage in a pharmacologic dose of TCDD (3.1 microM) or a control medium. The morphology was assessed, and staining for apoptosis was performed. Immunohistochemistry for the presence of aromatic hydrocarbon receptor (AhR) was performed in another set of morula-stage embryos. MAIN OUTCOME MEASURE(S) The number of embryos developing from the morula to the blastocyst stage and number of apoptotic blastomeres in control vs. TCDD culture conditions. RESULT(S) No statistically significant differences were observed in the percentage of embryos reaching the blastocyst stage: 80.9% (115 of 142) in the TCDD-treated group, vs. 82.9% (121 of 146) in the control group. There was also no difference in the degree of apoptosis: 22.6 +/- 7.3% apoptotic cells (TCDD) vs. 25.3 +/- 9.7% (controls). Staining indicated the slight presence of aromatic hydrocarbon receptor in the morula-stage mouse embryos. CONCLUSION(S) TCDD at 3.1 microM did not alter the development of early mouse morula to blastocysts and did not significantly induce apoptosis in vitro.

Elevated follicular fluid angiotensin II and pregnancy outcome

OBJECTIVE To assess whether a relationship exists between follicular fluid (FF) angiotensin II (AII) concentration and pregnancy outcome or earlier fecundity parameters and whether correlations exist among FF AII concentrations and P, E2, T, androstenedione (A), or various ratios of these. DESIGN Retrospective study in which hormone concentrations in FF samples were measured. SETTING In vitro fertilization clinic-Assisted Reproductive Technology Program, Rush Medical Center. PATIENTS Twenty-six female patients underwent ovarian stimulation for IVF. INTERVENTION Leuprolide acetate was combined with hMG and FSH for ovarian stimulation. MAIN OUTCOME MEASURE Follicular fluid aspirates were collected and oocytes were recovered 34 to 36 hours after hCG injection. The patients proceeded to undergo IVF and ET. Follicular fluid hormones were measured using standard RIA. Angiotensin II and steroid hormone concentrations in FF were compared for pregnant versus nonpregnant women using the Students t-test and rank-sum test. Pearson multiple-correlation analysis was performed to calculate correlation coefficients among AII concentrations and steroid concentrations in FF aspirates. RESULTS Mean FF concentration of AII was significantly lower in samples from women showing clinical pregnancies (112.2 +/- 13.9 pg/mL [107.3 +/- 13.3 pmol/L]) compared with samples from women who did not achieve pregnancy (217.1 +/- 23.8 pg/mL [207.5 +/- 22.7 pmol/L]) (mean +/- SE). A negative correlation was observed between FF concentrations of AII and P. Correlations of AII with E2, T, A, or with ratios of these did not show significance. CONCLUSION These data suggest that high AII concentration at time of oocyte recovery may indicate poor pregnancy outcome in women undergoing ovarian stimulation for IVF. These data corroborate previous results in animal models showing that AII predisposes follicles to undergo atresia-like conditions.

The effect of the oviduct, uterine, and in vitro environments on zona thinning in the mouse embryo

OBJECTIVE To evaluate the impact of the oviduct, uterine, and in vitro environments on zona pellucida thinning in the mouse embryo. DESIGN Female mice were stimulated with pregnant mare serum gonadotropin and mated and hCG injection. Unilateral oviduct ligation was performed on day 2 of gestation using the dorsal approach. The mice were divided into equal groups and killed on days 2, 3, 4, 5, and 10 of gestation. In vitro incubated embryos served as controls. Average daily zona thickness measurements were subjected to analysis of variance and paired Students t-test. SETTING The laboratory of the assisted reproductive program of Rush University Medical Center. MAIN OUTCOME MEASURE(S) Progressive daily decrease in average zona thickness. RESULT(S) Zona measurements of embryos flushed out of uterine horns, ligated oviducts, and in vitro incubation demonstrated statistically significant decreases in zona thickness, from 9.6 +/- 1.6 microns (day 3) to 6.0 +/- 0.8 microns (day 5), from 11.6 +/- 2.2 microns (day 2) to 6.0 +/- 1.6 microns (day 5), and from 11.1 +/- 2.0 microns (day 2) to 6.0 +/- 1.6 microns (day 5), respectively. There were no differences in average zona thickness for embryos in the same cell stage and same protocol day in all three locations. CONCLUSION(S) Zona thinning seems to be induced primarily by the dividing embryo before implantation. A substantial tubal and uterine contribution to zona thinning was not detected in this mouse embryo model.