Elizabeth S. Critser

Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics

Knowledge of bovine oocyte plasma membrane permeability characteristics at different developmental stages in the presence of cryoprotective agents (CPAs) is limited. The objective of this study was to determine the oolema hydraulic conductivity (Lp), cryoprotectant permeability (PCPA), and reflection coefficient (σ) for immature (germinal vesicle stage, GV) and in vitro–matured (metaphase II, MII) bovine oocytes. Two commonly used cryoprotective agents, dimethyl sulfoxide (DMSO) and ethylene glycol (EG), were studied. Osmometric studies were performed using a micromanipulator connected to an inverted microscope at 22 ± 2°C. Each oocyte was immobilized via a holding pipette, and osmotically induced volume changes over time (dv/dt) were recorded. The Lp values for GV and MII oocytes in DMSO (LpDMSO) were 0.70 ± 0.06 and 1.14 ± 0.07 μm/min/atm (mean ± SEM) and in EG (LpEG) were 0.50 ± 0.06 and 0.83 ± 0.07 μm/min/atm, respectively. Estimates of PDMSO for GV and MII oocytes were 0.36 ± 0.03 and 0.48 ± 0.03 μm/sec, and PEG values for GV and MII oocytes were 0.22 ± 0.03, 0.37 ± 0.03 μm/sec, respectively. The σ values for GV and MII oocytes in DMSO (σDMSO) were 0.86 ± 0.03 and 0.90 ± 0.04 and in EG (σEG) were 0.94 ± 0.03 and 0.76 ± 0.04, respectively. These data demonstrate that bovine oolema permeability coefficients to water and cryoprotectants change after in vitro maturation. Furthermore, the bovine oocyte PDMSO is higher than the PEG. These results may provide a biophysical basis for developing criteria for choosing optimal CPAs and for minimizing damage during addition and removal of the CPAs. Additionally, these data support the hypothesis that different procedures may be required for optimal cryopreservation of different oocyte developmental stages. Mol. Reprod. Dev. 49:408–415, 1998.

SALIVARY ANDROGEN-BINDING PROTEIN (ABP) MEDIATES SEXUAL ISOLATION IN MUS MUSCULUS

We wanted to determine whether the microevolution of the mouse salivary androgen‐binding protein (ABP) Alpha subunit gene (Abpa) could mediate sexual selection and thereby have a potential role in maintaining gene pool integrity where radiating mouse subspecies make secondary contact. This hypothesis is based upon previous work in this laboratory, which has shown that each subspecies apparently has its own allele and that these alleles have a 25‐fold excess of nonsynonymous/synonymous base substitutions compared to an average protein under purifying selection. We provide direct evidence for ABP‐assortative mate selection in a laboratory setting: Mus musculus domesticus and M. m. musculus female mice recognize and discriminate between the territories of male mice that essentially differ solely in their Abpa genotype and, when the males are present, the female prefers to mate with the one of her own ABP type. The observation that females could differentiate between the territories of the two males when those mice were absent suggests that the males marked their territories with ABP. In this study, we also detected ABP on the pelts of male mice and in their environment. It is likely that the animals apply the protein to their pelts by licking and that it is then deposited in their surroundings. We suggest that females of the two subspecies are able to discriminate between males of those subspecies on the basis of this protein molecule. Mouse salivary ABP might present a worthwhile system with which to study a prezygotic isolation mechanism in a mammal.

Antral follicles develop in xenografted cryopreserved african elephant (Loxodonta africana) ovarian tissue

The preservation of germ plasm from endangered species could augment captive breeding programs aimed at maintaining genetic diversity. Mammalian female germ plasm (oocytes) is extremely difficult to collect and cryopreserve; however, a promising alternative is the cryopreservation of ovarian tissue. In the present study, athymic nude (nu/nu) Balb/C mice were used to evaluate in vivo viability of cryopreserved ovarian tissue from Institute of Cancer Research genotype (ICR) mice or elephants. Female mice were ovariectomized prior to transplant of cryopreserved-thawed ovarian tissue from ICR mice (n=4) or elephants (n=6). Control mice were sham operated (n=4) or ovariectomized (n=5). Transplants were in the ovarian bursa, enabling in vivo ovulation and pregnancies from allografts. Vaginal cytology was monitored daily, and the intervals between and duration of epithelial cells present in smears were evaluated. Appearance of epithelial cells in sham-operated and allografted mice were at intervals of 4.3+/-0.6 and 3.3+/-0.5 days, lasting for 1.4+/-0.1 and 1.6+/-0.2 days, respectively. Sporadic incidence of epithelial cells in ovariectomized animals occurred at longer intervals (8.6+/-3.8 days). Females with xenografted elephant ovarian tissue demonstrated epithelial cells in vaginal smears at intervals of 4.5+/-1.0 days, for 2.5+/-0.5 days duration, which was significantly longer than the other groups (P < 0.05). Histological evaluation of tissues at the time of epithelial cells in smears demonstrated well-developed antral follicles, although oocytes were of poor morphological appearance or only cumulus-like complexes were seen. The nude mouse model is effective for assessing cryopreserved ovarian tissue xenograft function which can support the development of antral follicles.

Development of a novel microperfusion chamber for determination of cell membrane transport properties

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.

Cryobiology of Rat Embryos II: A Theoretical Model for the Development of Interrupted Slow Freezing Procedures

Abstract Current mammalian embryo cryopreservation protocols typically employ an interrupted slow freezing (ISF) procedure. In general, ISF consists of initial slow cooling, which raises the extracellular solute concentration, and results in cell dehydration. Permeating cryoprotective agents (CPAs), such as dimethyl sulfoxide (DMSO), are typically included in the medium to protect the cells against high solute concentrations. As this ISF procedure continues, slow cooling is terminated at an intermediate temperature (Tp), followed by plunging into liquid nitrogen (LN2). If the slow cooling step allowed a critical concentration ([CPA]c) of CPA to be reached within the cell, the CPA will interact with the remaining intracellular water during rapid cooling, resulting in the majority of the intracellular solution becoming vitrified and preventing damaging intracellular ice formation (IIF). This study presents a theoretical model to develop efficient ISF procedures, on the basis of previously developed data for the rat zygote. The model was used to select values of initial CPA concentrations and slow cooling rates (from initial estimated ranges of 0 to 4 molal DMSO and 0 to 2.5°C/min cooling rates) that would allow the intracellular solute concentration to exceed the critical concentration. The optimal combination was then determined from this range based on minimizing the duration of slow cooling.

Tissue Maturation in Vivo and in Vitro: Gamete and Early Embryo Ontogeny

Publisher Summary This chapter discusses various aspects of tissue maturation in vivo and in vitro. The primary oocyte stage of gamete differentiation in most female mammals is reached during fetal life after proliferation of primordial germ cells and oogonia with subsequent entry into meiosis and transformation of the gamete into the primary oocyte. Primary oocytes from tertiary follicles are most frequently recovered postmortem, surgically, with the aid of a laparoscope or most recently through ultrasound-guided follicular aspiration. Each in vitro protocol has its own rate of success and the outcome of the total process will be a multiplicative factor comprising all the steps. A system for producing bovine embryos in vitro has actually worked at some level of efficiency for a number of nondomestic ungulates. The buffering system in the medium selected for maturation will dictate the CO2 concentration required for culture. Macromolecular supplements used in tissue culture perform two basic roles. One of the roles is to provide a fixed source of nitrogen and the other is to reduce stickiness of the cell surfaces to make handling and manipulation easier. The follicular and ovarian factors affecting oocyte competency are also elaborated.

Evaluation of Circulating Anti-Sperm Antibodies in Fertile and Patient Populations

ABSTRACT: Several reports have demonstrated the presence of anti‐sperm antibodies (ASA) in infertile populations; however there is a paucity of information regarding ASA in fertile populations. The purpose of this study was to establish objective criteria for the interpretation of the Immuno‐bead Binding Test (IBT) based on values obtained from fertile individuals. Sera from 20 fertile couples (n = 40) were assayed by using a modification of the IBT previously described by Clarke et al. An initial lower limit of binding for positivity (lower limit) of 14% was used based upon the mean value for each isotype plus 2 standard deviations (SD) of 4 negative control sera assayed 4 to 7 times each. One‐way ANOVA or chi‐square analyses were used to analyze these data. There was no difference in percent immunobead binding between males and females in the fertile population (P > 0.1); therefore the data were pooled. Percent binding for fertile controls was: IgG, 21.7 + 31.9% (mean + SD); IgA, 19.5 + 25.8%; IgM, 16.9 + 14.9%. Initial analysis indicated no significant difference (P > 0.10) in percent binding between fertile and infertile individuals. The corresponding frequency of positive values (for at least one isotype) using a 14% lower limit was 23/40 (57.5%). This was not significantly different (P > 0.1) from the frequency observed in the patient population (140/ 242, 57.8%). New lower limits of positivity for each isotype were established based upon the mean plus 2 SD from the fertile control data: IgG, 85.4%; IgA, 71.1%; IgM, 46.7%. The frequencies of positive values obtained by using these criteria were not different (P > 0.1) between fertile and patient populations (20.0 vs. 18.7%, respectively). In addition, neither the number of beads bound per sperm nor the location of bead binding differed (P > 0.10) between the 2 populations. These data indicate that the frequency of circulating ASA is similar in fertile and infertile populations and suggests that ASA testing may be of questionable clinical significance.

Pregnancy resulting from peritoneal ovum sperm transfer procedure

This is the first reported transvaginal ultrasound-guided POST pregnancy in the United States. Advantages over the GIFT procedure include it being an office procedure done under local anesthesia and IV sedation, and decreased cost. Larger series are needed to compare pregnancy rates.

Pregnancy rates after peritoneal ovum-sperm transfer

We present the technique of peritoneal ovum-sperm transfer as an option for treatment in couples with unexplained infertility factors. In 1989 we reported the first successful pregnancy, in the United States, after transferring sperm and oocyte into the peritoneal cavity. We now report the results of a prospective study of this procedure. Twelve women with unexplained infertility underwent 23 cycles of peritoneal ovum-sperm transfer. Ovulation stimulation was achieved with human menopausal gonadotropin. Ultrasonographically directed oocyte recovery was performed by the transvaginal route with the patient under local anesthesia and sedation. After oocyte recovery, 4.5 +/- 0.4 (mean +/- SE) oocytes and 13.3 +/- 1.0 (mean +/- SE) x 10(6) motile spermatozoa were transferred into the pouch of Douglas. Six clinical pregnancies occurred in 23 stimulated cycles for a pregnancy rate of 26% per cycle. This value compares with the overall pregnancy rates of 16% for in vitro fertilization and 27% for gamete intrafallopian transfer reported by the In Vitro Fertilization Registry. Thus these preliminary data suggest that peritoneal ovum-sperm transfer is at least as successful as in vitro fertilization and gamete intrafallopian transfer. Advantages of peritoneal ovum-sperm transfer over gamete intrafallopian transfer include its being an office nonsurgical procedure not necessitating a general anesthetic and decreased cost. Therefore peritoneal ovum-sperm transfer is a reasonable first approach in couples with unexplained infertility.

Sperm antibodies after intraperitoneal insemination of sperm: a preliminary report.

ABSTRACT: To test the hypothesis that intraperitoneal insemination of sperm induces the expression of anti‐sperm antibodies a prospective study was designed. Fifteen women undergoing intraperitoneal insemination (with or without oocyte transfer) were studied with 11 women having evaluation of anti‐serm antibodies. Sperm antibodies were detected by the immunobead test prior to intraperitoneal insemination and after each treatment cycle. Two criteria were used to assess positivity: the first based upon negative controls and the second based upon the evaluation of 20 fertile control couples. Using the first criteria, of 11 of the women undergoing IPI for the first time, 7 were initially negative and 4 were initially positive for at least 1 isotype. After treatment, 3 additional patients were positive (for a total of 7) and 4 patients remained negative. This alteration in sperm antibody frequency was not different (P = .4) as determined by the Fishers Exact Test. Four of the 11 patients underwent a second cycle of IPI. All 4 patients were negative prior to the first treatment and 3 were negative prior to the second treatment. Subsequent to the second exposure, all 4 of these women were positive for at least 1 isotype. This shift in frequency distribution after 2 cycles was significant (P = .01). The frequency of antisperm antibodies for the same 11 patients was evaluated by using fertile control values as the basis of positivity. Two patients (18%) were positive for anti‐sperm antibodies prior to intraperitoneal insemination. There was no change in the frequency of positivity after 1 cycle of IPI. After 2 cycles of IPI 1 patient previously shown to be negative for anti‐sperm antibodies was subsequently positive. This shift in frequency was not significant (P = 0.1).