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Biomedical subjects

P Dandekar

Publications and source records attributed to P Dandekar.

8 recordsLinked to original sources

Perivitelline space of mammalian oocytes: extracellular matrix of unfertilized oocytes and formation of a cortical granule envelope following fertilization.

Extracellular matrices (ECM) present around unfertilized and fertilized mammalian oocytes were studied ultrastructurally in samples prepared in the presence of ruthenium red to facilitate stabilization of extracellular materials. Unfertilized mouse, hamster, and human oocytes have an ECM comprising granules and filaments in their perivitelline spaces (PVS). This matrix is more abundant in the human than in hamsters and mice. The granule/filament matrix appears identical to the matrix seen between cumulus and corona radiata cells following ruthenium red processing and previously shown to comprise protein and hyaluronic acid. By including ruthenium red during fixation, it is possible to demonstrate the existence of cortical granule exudate in the PVS of fertilized oocytes from hamsters, mice, and humans. Much of the cortical granule exudate is trapped in the PVS and forms a new coat around the fertilized oocyte. This material is particulate when stained with ruthenium red and appears to be uniformly dispersed around the entire oocyte surface. We refer to this new coat as the cortical granule envelope. This envelope is observed in the PVS of all developmental stages up to and including blastocysts in all three species. Following hatching of mouse and hamster blastocysts, the cortical granule envelope is no longer present. Possible functions of this envelope are discussed.

Animals

Structure, distribution and composition of the extracellular matrix of human oocytes and cumulus masses.

The structure, distribution and composition of the extracellular matrix present around the human oocyte and in the cumulus was examined following fixation in the presence of ruthenium red. An extracellular matrix comprising granules and filaments is present in the cumulus layer, in the corona radiata, in the outer pores of the zona pellucida and in the perivitelline space surrounding unfertilized oocytes. In replicate samples, the extracellular matrix comprised filaments which were mostly very long, occasionally cross-connected by shorter filaments, and usually decorated with numerous small granules. Enzymatic digestion with affinity-purified trypsin or Streptomyces hyaluronidase removes the granules and filaments, respectively, at all levels of the oocyte-cumulus complex. These results are interpreted to mean that protein and hyaluronic acid are present in all extracellular compartments of the human oocyte-cumulus complex. The significance of this distribution of hyaluronic acid with respect to the role of sperm hyaluronidase in fertilization is discussed.

Extracellular Matrix

Insulin-like growth factor I (IGF-I) levels in follicular fluid from human preovulatory follicles: correlation with serum IGF-I levels.

Insulin-like growth factor I (IGF-I) levels were measured in both serum and fluid of preovulatory follicles (n = 156) in 43 women undergoing in vitro fertilization (IVF). The mean IGF-I level in follicular fluid (FF) was significantly lower than in serum (0.52 +/- 0.02 IU/L versus 0.66 +/- 0.23 IU/L), and FF levels were significantly correlated with individual serum IGF-I levels as well as with follicular size and FF volume but not with oocyte maturity, granulosa cell appearance, or IVF. This suggests that FF IGF-I levels cannot serve as a clinical indicator for the degree of oocyte/granulosa cell differentiation or a predictor for IVF. Serum IGF-I levels were inversely correlated with the number of human menopausal gonadotropin ampules administered during treatment, suggesting that IGF-I might enhance ovarian gonadotropic stimulation.

Estradiol

Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells.

This report describes the detection and partial characterization of preovulatory human cumulus oophorus and mural granulosa cell-associated activity capable of initiating the human sperm acrosome reaction (AR) in vitro. Fragments of preovulatory human cumulus (cells plus extracellular matrix) were washed 3 times, incubated for 24 hr and the spent media and washes assayed for their ability to initiate the human sperm acrosome reaction (AR) in vitro. AR activity was present in the first two washes but not the third wash; however, AR activity was recovered in the spent medium after 3 X-washed fragments were incubated for 24 hr under conditions which maintained the viability of the cumulus cells. The spent media of preovulatory human mural granulosa cells contained AR-initiating activity after 1-3, 3-6, and 6-9 days of culture. The properties of the AR activity present in spent media of human cumulus fragments included resistance to loss of activity during treatment with pronase; resistance to loss of activity during treatment with chondroitinase ABC or bacterial hyaluronidase; heat stability after overnight incubation; lack of extraction by chloroform-methanol; an apparent molecular weight (MW) of 50,000, as determined by Sephadex G-75 column chromatography; conversion to a lower apparent MW activity by incubation with pronase. These properties are also characteristic of a fraction derived by Sephadex G-75 chromatography of preovulatory human follicular fluid which also has been shown to stimulate the human sperm acrosome reaction in vitro. The AR activity from spent media of human mural granulosa cells is also found in a 50,000 MW Sephadex G-75 fraction. We propose that the sources of the 50,000 MW human follicular fluid AR activity are the cumulus oophorus and the mural granulosa cells.

Acrosome

A comparison of in-vitro fertilization of rabbit eggs using spermatozoa recovered from the uterus or vagina.

Spermatozoa were recovered from the uterine horns or vagina of mated rabbit does 12-20 hr p.c. and their fertilizing capacity tested in an in-vitro system. Uterine spermatozoa gave consistently high fertilization (93-100%) throughout. Vaginal spermatozoa gave good results (81-85%) 12-14 hr p.c. but fertilization then declined with time. Vaginal spermatozoa recovered from mated females with or without ligation of the uterine horns were equally able to fertilize eggs. Fragmentation of uncleaved eggs was noted in experiments with vaginal spermatozoa, but never with uterine spermatozoa. The former were slower in penetrating eggs and the zygotes were correspondingly slower in cleaving. Triploidy, possibly due to ageing, was found in approximately 15% of the embryos fertilized by vaginal spermatozoa.

Animals

Localization of prostaglandin on the plasmalemma of rabbit sperm.

Prostaglandin and prostaglandin receptors were localized on unwashed ejaculate rabbit sperm utilizing ferritin conjugated antibody to prostaglandin. Reaction was obtained over the acrosomal portion of the plasma membrane, with a greater aggregation over the apex of the head. The remainder of the head plasmalemma and flagellum showed no deposits of of reaction product. The reaction did not occur if the sperm were washed prior to incubation with the labelled antisera. However enhanced binding of ferritin was demonstrated if washed sperm were pre-incubated in PGE1. No reaction was seen in similar studies with human sperm.

Animals