Live birth after ovarian tissue transplant.

Radiation and high-dose chemotherapy may render women with cancer prematurely sterile, a side-effect that would be avoided if ovarian tissue that had been removed before treatment could be made to function afterwards. Live offspring have been produced from transplanted ovarian tissue in mice and sheep but not in monkeys or humans, although sex steroid hormones are still secreted. Here we describe the successful transplantation of fresh ovarian tissue to a different site in a monkey, which has led to the birth of a healthy female after oocyte production, fertilization and transfer to a surrogate mother. The ectopically grafted tissue functions without surgical connection to major blood vessels and sets the stage for the transplantation of cryopreserved ovarian tissue in humans.

The future of human ovarian cryopreservation and transplantation: fertility and beyond.

OBJECTIVE: To review the current progress in ovarian cryopreservation and transplantation and to discuss the obstacles with the clinical application of this technique. DESIGN: The literature on ovarian cryopreservation and transplantation was reviewed to facilitate understanding and predict future directions. The studies related to this topic were identified through MEDLINE and other bibliographic databases, focusing on the most recent developments. CONCLUSION(S): The experimental evidence for low-temperature storage of ovarian tissue is encouraging. Although restoration of fertility with cryopreserved ovarian grafts has been successful in various animals, there are uncertainties about the optimum use of stored ovarian tissue in humans. Autotransplantation appears to be promising, but the potential risk of transmitting malignant cells in women with cancer is of great concern. The maturation of primordial follicles with xenotransplantation is encouraging, but the efficacy and the safety of this method need further investigation. Furthermore, the quality of oocytes that have been matured in a host animal is unknown. The development of in vitro culture systems for oocyte maturation is still in its infancy. There are many issues to be resolved in ovarian transplantation before the full clinical use of this emerging technique. Most of all, there is an urgent need to optimize the freeze/thaw procedure and to find the means to protect grafts from ischemia-reperfusion injury. Nevertheless, ovarian transplantation should prove to be clinically useful for women at risk for premature ovarian failure.

Ovarian follicular concentrations of activin, follistatin, inhibin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3, and vascular endothelial growth factor in spontaneous menstrual cycles of normal women of advanced reproductive age.

Previous studies indicate that the menstrual cycles of older reproductive age women are characterized by a selective elevation of FSH associated with early development and ovulation of a dominant follicle. Several intraovarian hormones and growth factors have been identified that appear to serve important paracrine roles. The purpose of this study was to examine follicular fluid (FF) hormones and growth factors in the dominant follicle of unstimulated cycles of older, ovulatory women. We aspirated FF from the preovulatory dominant follicle in natural menstrual cycles of older subjects (age, 40-45 yr; n = 20) and younger controls (age, 20-25 yr; n = 19). FF was analyzed for estradiol, progesterone, testosterone, androstenedione, inhibin A and B, total activin A, total follistatin, insulin-like growth factor I (IGF-I), IGF-II, IGF-binding protein-2 (IGFBP-2), IGFBP-3, and vascular endothelial growth factor (VEGF) concentrations. We found that the dominant follicles from older women contain normal concentrations of steroids, inhibin A and B, IGF-II, IGFBP-2, and IGFBP-3; increased concentrations of follistatin, activin A, and VEGF; and decreased concentrations of IGF-I. Therefore, under the influence of elevated FSH, the dominant follicle in older women is highly competent in terms of hormone and growth factor secretion. We postulate that elevated FF activin may be related to the early ovulation observed in older women, whereas elevated VEGF may be related to the meiotic spindle abnormalities observed in the oocytes of older reproductive age women.

Morphometric analysis of primordial follicle number in pigtailed monkey ovaries: symmetry and relationship with age.

We previously described a modern, three-dimensional counting method for determining primordial follicle (PF) numbers in primate ovaries using a combination of fractionator and physical dissector techniques. The purposes of our current study were 1) to apply our method to describe intraindividual differences in PF numbers between ovaries and 2) perform a linear regression analysis of age versus mean PF number per ovary. Ovaries from 16 pigtailed monkeys (Macaca nemestrina) age 0.85-12.5 yr were examined. Both ovaries were available from 11 subjects. The difference between ovaries ranged from 2% to 22% (mean +/- SD, 10 +/- 7%) and was not statistically significant. Regression analysis of data from all 16 subjects displayed a log-linear relationship according to the equation log N(a) = 4.8542 - 0.0714(age) where N(a) is the number of PF at a given chronological age. The fit for this model was highly significant (r(2) = 0.73, p

Inhibin and reproductive aging in women.

The monotropic FSH rise is the sentinel endocrine event that first indicates a woman is approaching the end of her reproductive potential. While a deficiency in inhibin has long been postulated as the immediate cause of the monotropic FSH rise, this has only recently been demonstrated to actually occur. It is our current hypothesis that when the number of preantral follicles in both ovaries drop below a threshold, then there is a subtle decrease in inhibin B which leads to the monotropic FSH rise which, in turn, accelerates follicular depletion and the attainment of the menopause.

Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm.

OBJECTIVE: To examine the outcome of intracytoplasmic sperm injection (ICSI) with round-headed sperm (globozoospermia). DESIGN: Retrospective analysis. SETTING: In vitro fertilization laboratory with extensive ICSI experience. PATIENT(S): A patient couple with infertility because of globozoospermia seeking ICSI treatment. MAIN OUTCOME MEASURE(S): Fertilization, cleavage, and pregnancy rates. INTERVENTION(S): Intracytoplasmic sperm injection and calcium ionophore. RESULT(S): This couple experienced only 7% fertilization after ICSI in their first cycle. Treatment of the unfertilized oocytes with calcium ionophore 20 hours after ICSI-induced fertilization and cleavage of 70% of the oocytes. Embryo quality was fair to good. On the second cycle, 8 of the injected oocytes were treated with ionophore immediately after ICSI and the remaining 20 oocytes were untreated. Normal fertilization was achieved in 75% of the treated and 10% of the untreated oocytes. Treatment of these unfertilized oocytes with ionophore 20 hours after ICSI resulted in fertilization in 73%. Pregnancy was not achieved after either ICSI cycle. Ultrastructural analysis indicated multiple structural abnormalities in the sperm. CONCLUSION(S): These results indicate that the round-headed sperm from this patient were incapable of oocyte activation after ICSI. This may be the reason for the frequent ICSI fertilization failure seen with this condition. Current ICSI procedures may not always overcome the infertility associated with globozoospermia, and further study of the etiology of this condition is needed.

An accurate, simple method for unbiased determination of primordial follicle number in the primate ovary.

Previous investigations of primordial follicle (PF) number in primate ovaries have used biased, model-based techniques that require correction factors based on assumptions regarding cell size, orientation, and shape. We sought to apply several techniques from the "new stereology" to obtain unbiased number estimates. This method involves a hierarchy of systematic random sampling combined with the physical disector and fractionator techniques. The method readily allows the estimation of the coefficient of error (CE) of each sampling level as it contributes to the observed variance of the overall number estimate. We examined one ovary from each of five pigtailed monkeys (Macaca nemestrina). The mean number of PF was 15,735 +/- 6214 (mean +/- SD). The mean CE for the individual number estimates was 0.085, contributing minimally to the inter-individual coefficient of variation (CV) of the primordial follicle numbers (CV = 0.395). The correlation between age and PF number was not significant (r = -0.74, p > 0.1). The total time taken to count the 100-200 PF necessary per ovary was 4-5 h. We conclude that this method produces reliable, unbiased estimates with measurable and acceptable accuracy in a robust, efficient manner.

Changes in centrosomal domains during meiotic maturation in the human oocyte.

The distribution of microtubule organizing centres (MTOC) in the human oocyte was examined using the microtubule-active drug, taxol, to promote polymerization. Oocytes were obtained from gonadotrophin-stimulated in-vitro fertilization (IVF) patients and examined during various phases of meiotic maturation using confocal fluorescence microscopy. During the prophase of meiosis I, taxol failed to stimulate microtubule nucleation in any region of the cells. Only a few microtubules were visible in the oocyte cortex. As the transition from prophase to metaphase began, during germinal vesicle breakdown, taxol stimulated the appearance of a small number of isolated aster-like arrays of microtubules in the cortex, predominantly in regions adjacent to the nucleus. Oocytes treated with taxol when they had reached the first meiotic metaphase exhibited a large number of aster-like microtubule arrays. These structures were predominantly located in the cortical region of the oocyte, but smaller arrays were also visible in the endoplasmic areas. There did not appear to be any increased density of asters in any particular cortical or endoplasmic region. Oocytes treated with taxol during the second metaphase of meiosis exhibited a similar response to the drug as seen during metaphase I, Again, the microtubule asters were most prevalent in the cortex, with smaller, less dense arrays in the endoplasm. The metaphase spindle was also affected by taxol as revealed by increased density and hyperelongation of microtubules at the poles of the spindle as compared to untreated oocytes. The metaphase plate of chromosomes was highly disrupted by taxol treatment, most likely due to the forces generated by microtubule elongation. We conclude that the human oocyte develops MTOC as meiotic maturation proceeds beyond the prophase I arrest. The first MTOCs are perinuclear, but the number and distribution increases widely as the oocytes enter metaphase. We hypothesize that the centrosome of the human recruits several MTOC domains for the assembly of the meiotic spindles in both meiotic divisions. It is speculated that one or more of the non-spindle-associated MTOCs may combine with sperm centrosomal material during fertilization to create the complete centrosome needed for embryonic mitosis. The widespread distribution of MTOC foci throughout the cortex may ensure this recombination regardless of the point of sperm incorporation into the oocyte.

Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women.

To examine the effects of maternal ageing on the meiotic apparatus, we obtained oocytes from naturally cycling women in two age groups, including younger (aged 20-25 years) and older (aged 40-45 years) women. Using high-resolution confocal microscopy we obtained a detailed picture of the meiotic spindle and chromosome placement during various phases of meiosis. Our data revealed that the meiotic spindle in older women is frequently abnormal, both with regard to chromosome alignment and the microtubule matrix that comprise the meiotic spindle. The spindle in 79% of the oocytes from the older group exhibited abnormal tubulin placement and one or more chromosomes were displaced from the metaphase plate during the second meiotic division. In contrast, only 17% of the oocytes from the younger age group exhibited aneuploid conditions. The majority of eggs from this group possessed a well ordered, meiotic spindle containing chromosomes that were fully aligned within a distinct metaphase plate in the spindle. Chromosome management during meiosis is directed by microtubule assembly within the spindle. These data suggest that the regulatory mechanisms responsible for assembly of the meiotic spindle are significantly altered in older women, leading to the high prevalence of aneuploidy.

Decreased inhibin B secretion is associated with the monotropic FSH rise in older, ovulatory women: a study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles.

UNLABELLED: This study sought to compare circulating and follicular fluid (FF) concentrations of dimeric inhibin A and B utilizing specific two-site ELISAs for these hormones in normal older and younger ovulatory women. METHODS. Normally ovulating women age 40-45 (n = 10) and 20-25 (n = 13) were studied throughout the follicular phase with daily blood sampling, transvaginal ultrasound examinations, and dominant follicle aspiration. When the dominant follicle reached a mean diameter of 16 mm or serum estradiol (E2) was > or = 550 pmol/L, 10,000 IU of hCG was administered intramuscularly followed 32 hours later by transvaginal follicle aspiration. Serum and FF samples were analyzed for E2, FSH, and inhibin A and B. Daily hormone levels were compared by ANOVA, and mean results were compared using t-tests. RESULTS: Older women developed a dominant follicle sooner, meeting criteria for hCG cycle day 10.6 +/- 0.4 vs. 14.5 +/- 1.0 p < 0.001. As expected, the older group had higher maximal serum FSH concentrations compared to the younger women (11.4 +/- 0.5 vs. 8.0 +/- 0.4 IU/L, p < 0.001). We compared hormone concentrations from days-1 to 3 (where day 0 = day of maximal FSH concentration). E2 concentration was higher in the older women (p = 0.002), and there was no significant difference in inhibin A secretion (p = 0.61). In contrast, mean inhibin B concentration was significantly lower in the older women (p = 0.04). On the day of aspiration of the dominant follicle, serum inhibin B was decreased in the older subjects (42.6 +/- 6.5 vs. 153.1 +/- 53 pg/ml, p = 0.02), whereas older subjects had higher levels of inhibin A (106 +/- 16 vs. 60.4 +/- 9.4 pg/ml, p = 0.04) and similar E2 levels (665 +/- 35.2 vs. 687 +/- 92 pmol/L, p = 0.83). There were no differences in FF concentrations of inhibin B (164 +/- 31 vs. 174 +/- 37 ng/ml, p = 0.85), inhibin A (317.7 +/- 38 vs. 248 +/- 57 ng/ml, p = 0.16), or E2 (2074 +/- 294 vs. 2474 +/- 338 nmol/L, p = 0.82) in the older and younger women. CONCLUSION. Follicular phase inhibin B secretion is decreased in older ovulatory women who demonstrate a monotropic FSH rise, whereas inhibin A secretion is similar to that in younger women. The dominant follicle in these older women appears to be normal in terms of FF E2 and inhibin content. We speculate that decreased inhibin B secretion most likely reflects a diminished follicular pool in older women and may be an important regulator of the monotropic FSH rise.

Ovarian follicular development and the follicular fluid hormones and growth factors in normal women of advanced reproductive age.

Reproductive aging in women (a physiological decline in the function of the hypothalamic-pituitary-ovarian axis) is an infrequently investigated and poorly understood biological phenomenon. Although menstrual irregularity and anovulation are known to precede the menopause, normal women in their fifth decade experience a profound decrease in fertility while still experiencing regular menstrual cycles. To further our understanding of the physiological changes associated with reproductive aging, this study examined the spontaneous development and function of ovarian follicles in normal women, aged 40-45 yr. The subjects were women (n = 21), aged 40-45 yr, who had regular 25- to 35-day ovulatory menstrual cycles, were not infertile, had no medical problems, and met specific criteria for weight, diet, and exercise. The controls were normal women (n = 20), age 20-25 yr, who met the same criteria. The subjects were monitored with daily hormone measurements [LH, FSH, estradiol (E), progesterone (P), and inhibin] and pelvic sonograms from day 1 of their study cycle until the dominant ovarian follicle reached a mean diameter of 15 mm and/or a serum E level of 550 pmol/L or higher was attained. At that time, 10,000 U hCG were given, and a transvaginal sonographic follicle aspiration was performed 32 h later. The follicular fluid (FF) was collected, stored frozen at -70 C, and later analyzed for E, P, testosterone (T), androstenedione, inhibin, insulin-like growth factor I (IGF-I), and IGF-II. The number of cycle days to aspiration was lower (11.6 vs. 15.6 days; P < 0.001) and the early follicular phase mean FSH and mean E levels were higher (9.3 vs. 6.6 mIU/mL and 305 vs. 160 pmol/L; P < 0.01) in the older (O) group compared to the younger group. There was a strong trend toward higher FF mean E (2280 vs. 1931 nmol/L) and lower FF mean T (978 vs. 2361 pmol/L) levels in group O. The E/T ratio was significantly higher (5253 vs. 2408; P < 0.03) in group O. In group O, the mean FF P levels were increased as well (25.1 vs. 18.8 micromol/L; P < 0.01). The serum mean IGF-I (153 vs. 226 ng/mL; P < 0.001) and FF mean IGF-I (113 vs. 158 ng/mL; P < 0.02) levels were significantly decreased in group O. There were no differences between groups in serum or FF IGF-II or inhibin levels. Whether reproductive aging is an intrinsic ovarian process or the ovary is simply responding to exogenous influences, the ovary in general and its follicles in particular are the primary site of the effects of aging. Ovarian follicles in older ovulatory women have some unique features: 1) the follicles are the same size as those in younger women, but form more rapidly; 2) secretion of E and inhibin is not compromised; 3) the concentrations of steroids in the FF are indicative of a healthier follicle, i.e. increased P levels and higher estrogen to androgen ratio; and 4) serum and FF levels of IGF-I are decreased, but there are no differences in IGF-II levels.

The anterior pituitary response to a gonadotropin-releasing hormone challenge test in normal older reproductive-age women.

OBJECTIVE: To assess the pituitary responsiveness to GnRH stimulation of premenopausal women relative to age. DESIGN: Older and younger reproductive-age women underwent the GnRH stimulation test in the early follicular phase of the menstrual cycle. SETTING: Female subjects in an academic research environment. PATIENTS: Women aged 21 to 44 years consisting of normal volunteers and infertility patients. INTERVENTIONS: Gonadotropin-releasing hormone was administered intravenously between days 2 and 4 of the menstrual cycle. Blood samples were collected from -20 minutes before to 120 minutes after administration. MAIN OUTCOME MEASURE: Luteinizing hormone, FSH, inhibin, and E2 levels. RESULTS: No significant difference in baseline values existed between older and younger women with regard to LH, inhibin, and E2, but basal FSH levels were higher in older women. A significantly diminished percent of LH and percent FSH change above baseline occurred 30 minutes after GnRH administration in the older women compared with younger women. No change in inhibin or E2 levels could be detected during the sampling period. CONCLUSIONS: The present study demonstrates marked attenuation of the acute pituitary LH response (sensitivity) to GnRH stimulation in older women when compared with a younger cohort.

Reproductive aging: accelerated ovarian follicular development associated with a monotropic follicle-stimulating hormone rise in normal older women.

Women experience a decline in fertility that precedes the menopause by several years. Previous studies have demonstrated a monotropic rise in FSH associated with reproductive aging: however, the mechanism of this rise and its role in the aging process are poorly understood. The purpose of this study was to characterize ovarian follicular development and ovarian hormone secretion in older reproductive age women. Sixteen women, aged 40-45 yr, with regular ovulatory cycles were studied. The control group consisted of 12 ovulatory women, aged 20-25 yr. Serum obtained by daily blood sampling was analyzed for FSH, LH, estradiol (E), progesterone, and inhibin (Monash polyclonal assay). Follicle growth and ovulation were documented by transvaginal ultrasound. Older women had significantly higher levels of FSH throughout the menstrual cycle. E, progesterone, LH, and inhibin levels did not differ between the two age groups when compared relative to the day of the LH surge. Ultrasound revealed normal growth, size, and collapse of a dominant follicle in all subjects. Older women had significantly shorter follicular phase length associated with an early acute rise in follicular phase E, reflecting accelerated development of a dominant follicle. We conclude that older reproductive age women have accelerated development of a dominant follicle in the presence of the monotropic FSH rise. This is manifested as a shortened follicular phase and elevated follicular phase E. The fact that ovarian steroid and inhibin secretion were similar to those in the younger women suggests that elevated FSH in women of advanced reproductive age may represent a primary neuroendocrine change associated with reproductive aging.

Circulating levels of growth hormone, insulin-like growth factor-I and growth hormone binding protein in normal women of advanced reproductive age.

OBJECTIVE: Women experience an age-related decline in fertility despite regular ovulatory cycles and normal production of ovarian steroids. Growth hormone and IGF-I are both reported to decline with age, and there is evidence that both hormones promote intraovarian actions of gonadotrophins. The purpose of this study was to characterize circulating levels of GH and IGF-I in normal, older reproductive age women with ovulatory cycles. DESIGN: Prospective, controlled. PATIENTS: Twenty-eight regularly cycling older (n = 16) and younger (n = 12) women were recruited for daily blood sampling throughout a menstrual cycle. MEASUREMENTS: Serum obtained from daily blood sampling was analysed for LH, FSH, oestradiol (E2) and progesterone (P). Serum obtained from frequent sampling during the admission was analysed for pulsatile GH secretion. IGF-I and GH binding protein (GHBP) were also measured in subsets of the two age groups. RESULTS: All subjects exhibited normal patterns of LH, FSH, E2 and P consistent with ovulatory cycles. There were no differences between the two age groups in integrated 24-hour GH secretion or in GH pulse amplitude or frequency. There were no differences in GH secretion between the early follicular and miduluteal phases when data were combined for the two subject groups. Plasma concentrations of IGF-I were significantly lower throughout the cycle in the older women. There were no significant differences in levels of GHBP across the cycle or between the two age groups. CONCLUSIONS: IGF-I decreases with age in women without identifiable changes in the amount or pattern of GH secretion or in circulating GHBP concentrations. Decreased IGF-I production may be related to decreased ovarian gonadotrophin sensitivity in older reproductive age women.

The gonadotropin secretion pattern in normal women of advanced reproductive age in relation to the monotropic FSH rise.

OBJECTIVE: Women of advanced reproductive age are known to demonstrate subtle FSH elevations (monotropic FSH rise) while still retaining ovulatory function. The purpose of this study was to investigate the hypothesis that the physiologic basis for the monotropic FSH rise is an alteration in the secretion pattern of the GnRH pulse generator. METHODS: The subjects were 11 normal women age 40-45 years who underwent 24 hours of frequent blood sampling in the follicular (EF) and/or midluteal (ML) phases of spontaneous menstrual cycles. The controls were 11 normal women age 20-25 years. The respective gonadotropin secretion patterns were analyzed for LH pulse frequency, mean LH and FSH levels, and LH pulse amplitude. RESULTS: There were no differences between the groups for estradiol (E2) and progesterone when the respective cycle phases were compared. The 24-hour mean FSH level was significantly increased in the older women in both the EF and ML phases. There were no differences between the groups in either cycle phase for LH pulse frequency, LH pulse amplitude, and mean LH levels. CONCLUSION: The results lend no support to the hypothesis that a slowing or other alteration of the GnRH pulse generator is the basis for the monotropic FSH rise in older ovulatory women. Other possibilities include the dynamics of E2 secretion or changes in FSH-modulating peptides (i.e., inhibin) in these women.

Early persistent activation of sperm K+ channels by the egg peptide speract.

Transduction by sperm of the instructive signal provided by the egg peptide speract involves rapid, complex changes in internal ion and cyclic nucleotide content. Here, investigations of hypotonically swollen sperm provide insight into the underlying processes and identify K+ channel activation as an initial ionic event in gamete recognition. A sustained hyperpolarization of swollen sperm is promoted by less than 2.5 pM speract and is followed (with greater than 100 pM speract) by transient repolarization and (with greater than 10 nM speract) by depolarization that is dependent on external Ca2+. Monophasic increases in pHi are produced only by greater than 25 pM speract, indicating that hyperpolarization may not directly promote alkalinization. Increased K(+)-selective (K+ greater than Rb+ greater than Cs+ greater than Na+) membrane permeability is found after all speract greater than 2.5 pM, suggesting that hyperpolarization results from persistent activation of K+ channels and that repolarization has a different ionic basis. Supporting this contention, the K+ channel blocker tetraethylammonium (20 mM) inhibits the increased K+ permeability that follows treatment of swollen sperm (and of sperm in seawater) with 2.5 pM speract. Such induced activation of K+ channels is observed in patch-clamped swollen sperm examined in the cell-attached configuration, upon application of 5-50 pM speract to the bath medium. The efficacy of externally applied speract and its potency indicate that activation is indirect and probably involves an as yet unidentified diffusible mediator whose production is promoted by speract at concentrations 0.01-0.001 times those predicted from reported estimates of the Kd for the known speract receptor.

Localization and developmental fate of ovoperoxidase and proteoliaisin, two proteins involved in fertilization envelope assembly.

Fertilization of the sea urchin egg leads to the assembly of an extracellular matrix, the fertilization envelope. Ovoperoxidase, the enzyme implicated in hardening the fertilization envelope, is inserted into the assembling structure via a Ca2+-dependent interaction with the protein proteoliasin (P. Weidman and B. M. Shapiro, 1987, J. Cell Biol. 105, 561-567). In the present report, polyclonal antisera were raised to ovoperoxidase and proteoliasin (purified from eggs of Strongylocentrotus purpuratus) and characterized by Western blot analysis and an enzyme-linked immunoabsorbent assay (ELISA). By indirect immunofluorescence microscopy all cortical granules of unfertilized eggs, as well as the fertilization envelope, contained both proteoliasin and ovoperoxidase. At the ultrastructural level both proteins are localized to the electron-dense spiral lamellae of the cortical granules. Western blot analysis revealed that ovoperoxidase and proteoliasin persist in early embryos until hatching, but are absent from later developmental stages. Homogenates of eggs of several other echinoderm species (Strongylocentrotus droebachiensis, Strongylocentrotus franciscanus, Pisaster ochraceus, Dendraster excentricus, and Lytechinus pictus) also contain proteins antigenically similar to ovoperoxidase and proteoliaisin, indicating that many echinoderms utilize a similar strategy for assembly of the fertilization envelope. The results underline the need for postsecretory controls in the extracellular matrix modifications that accompany the cortical reaction.

Hierarchies of protein cross-linking in the extracellular matrix: involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly.

The involvement of transglutaminase activity in fertilization envelope (FE) formation was investigated using eggs from the sea urchin, Strongylocentrotus purpuratus. Eggs fertilized in the presence of the transglutaminase inhibitors, putrescine and cadaverine, had disorganized and expanded FEs with inhibition of the characteristic I-T transition. The permeability of the FE was increased by these agents, as revealed by the loss of proteins from the perivitelline space and the appearance of ovoperoxidase activity in supernates from putrescine-treated eggs. [3H]putrescine was incorporated into the FE during fertilization in a reaction catalyzed by an egg surface transglutaminase that could also use dimethylcasein as a substrate in vitelline layer-denuded eggs. Egg secretory products alone had no transglutaminase activity. The cell surface transglutaminase activity was transient and maximal within 4 min of activation. The enzyme was Ca2+ dependent and was inhibited by Zn2+. We conclude that sea urchin egg surface transglutaminase catalyzes an early step in a hierarchy of cross-linking events during FE assembly, one that occurs before ovoperoxidase-mediated dityrosine formation (Foerder, C. A., and B. M. Shapiro. 1977. Proc. Natl. Acad. Sci. USA. 74:4214-4218). Thus it provides a graphic example of the physiological function of a cell surface transglutaminase.

Influence of the calcium ionophore A23187 on rat egg behavior and cortical F-actin.

Rat eggs treated with the calcium ionophore A23187 and subjected to long-term observation by phase microscopy were found to undergo many developmental changes that are normally associated with fertilization. These included cortical granule exocytosis and the abstriction of the second polar body. In addition, time-lapse video microscopy revealed that, unlike untreated eggs, whose surfaces remained relatively immotile, the ionophore-treated eggs underwent a lengthy period of surface undulatory activity. Since all of these events were remarkably similar in timing and morphology to those seen in fertilized eggs, we conclude that A23187 is capable of activating rat eggs. Using NBD-phallacidin, the distribution of F-actin in ionophore-activated eggs was determined. During most of the postactivation period the eggs possessed an uninterrupted, uniform band of polymerized actin encompassing the entire cortex of the egg. However, during a discrete 1.5-h period after the formation of the second polar body, an area adjacent to the region of polar body abstriction exhibited more intense staining than the rest of the cortex. Cytochalasin B treatment caused a dramatic reduction and/or rearrangement in cortical NBD-phallacidin staining in activated eggs as compared to activated controls not exposed to the drug. We observed that all the developmental changes described above could be produced in the absence of exogenous calcium, suggesting that the rat egg possesses internal stores of calcium sufficient to elicit an activational response. We conclude that the ionophore-induced release of free calcium ions into the cytosol stimulates many of the developmental changes that are normally seen during fertilization. These results indicate that calcium influx and cytoskeletal activity are correlated during the activation of this animal egg.

The distribution of polymerized actin in the rat egg and its sensitivity to cytochalasin B during fertilization.

The distribution of polymerized actin in rat eggs fertilized in vitro was determined using NBD-phallacidin (NBD-ph). Unfertilized and fertilized eggs exhibited a 3-5-micron-thick band of fluorescence that encompassed the entire cortical cytoplasm. There was no dramatic increase in the staining of the cortex in association with any component of the fertilizing sperm during its incorporation into the egg. Unfertilized eggs and fertilized eggs obtained at intervals after sperm-egg fusion were treated with cytochalasin B (CB; 5 micrograms/ml) and subsequently stained with NBD-ph. Unfertilized eggs treated with CB exhibited a continuous ring of cortical staining identical to that seen in untreated eggs. Eggs treated with CB 15 min after sperm-egg fusion exhibited small gaps in the cortical staining pattern, whereas those exposed to CB 1 hr after fusion exhibited larger gaps and the staining pattern appeared punctate. This pattern could be seen throughout the remainder of the 7 hr period of sperm incorporation and for at least 13 hr thereafter. CB-treated fertilized eggs that were washed to remove the drug again exhibited uninterrupted cortical staining on treatment with NBD-ph. CB also induced the resorption of surface elevations that are normally seen on the eggs during sperm incorporation, but it did not affect the morphology of unfertilized eggs. The sensitivity to CB during fertilization coincides with the onset of a variety of egg shape changes that occur during the period of sperm incorporation (Battaglia and Gaddum-Rosse, Gamete Res., 10:107-118, 1984a).(ABSTRACT TRUNCATED AT 250 WORDS)