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  • Author: PRAMILA DANDEKAR x
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Pramila Dandekar and Lynn Fraser

Summary.

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.

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Mildred Gordon and Pramila V. Dandekar

The mammalian spermatozoon undergoes a series of maturational changes after leaving the testis. Spermatozoa in the caput epididymidis are not fertilizable (Orgebin-Crist, 1967) but are functional when they reach the cauda. Differentiation of the membranes of caput spermatozoa during epididymal transit was demonstrated by the binding of colloidal iron (Cooper & Bedford, 1971) and Concanavalin A (Gordon, Dandekar & Bartoszewicz, 1975). Exposure to seminal plasma appears to stabilize the surface coat (Gordon et al., 1975), the plasma membrane enzymes (Gordon, 1973), and acrosomal enzymes (Zaneveld, Dragoje & Schumacher, 1972). Spermatozoa become capacitated in the female genital tract, a process which removes seminal substances (Ericsson, 1967; Oliphant & Brackett, 1973) and modifies the lectin binding of the membrane (Gordon et al., 1975). The segment of the plasma membrane overlying the acrosome of spermatozoa from the cauda epididymidis contains a neutral ATPase which is not active in ejaculates (Gordon, 1973) and it was postulated that the enzyme may become competent during capacitation following removal of seminal antigens.

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LYNN R. FRASER and PRAMILA V. DANDEKAR

To achieve fertilization of mammalian eggs in vitro, the gametes are often mixed and incubated in a CO2-enriched atmosphere, e.g. rabbit (Brackett & Williams, 1968), mouse (Cross & Brinster, 1970), human (Edwards, Steptoe & Purdy, 1970). Since the various media are usually buffered with relatively high levels of bicarbonate, the CO2 is needed to maintain a pH which will allow sperm penetration. Bicarbonate has a stimulatory effect on sperm metabolism (Murdoch & White, 1971) and may facilitate capacitation of hamster spermatozoa in vitro (Bavister, 1969). Furthermore, replacement of bicarbonate with tris or phosphate in a defined medium for ovum culture resulted in cessation of development (Brinster, 1969).

Using the technique of Brackett & Williams (1968) in which eggs and spermatozoa are cultured for 4 hr in an atmosphere of 5% CO2:95% air, it has been possible to achieve a high rate of fertilization of rabbit eggs

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Lynn R. Fraser and Pramila V. Dandekar

Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut, U.S.A.

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RAMA A. VAIDYA, R. H. GLASS, PRAMILA DANDEKAR and K. JOHNSON

Since the introduction of the concept of capacitation, numerous investigators have attempted to define the changes that spermatozoa undergo in the female reproductive tract. Studies utilizing light and electron microscopy have failed to show alterations in sperm structure following incubation in the rabbit uterus for periods up to 15 hr, a length of time sufficient to accomplish capacitation (Bedford, 1970). The loss of tetracycline fluorescence from spermatozoa in the oestrous uterus has been suggested as an indicator of capacitation (Ericsson, 1967), but it has since been shown conclusively that this theory can no longer be considered tenable (Vaidya, Bedford, Glass & Morris, 1969).

Changes in the net negative surface charge of rabbit spermatozoa during passage through the epididymis have been demonstrated by micro-electrophoresis (Bedford, 1963). This communication describes changes

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LYNN R. FRASER, PRAMILA V. DANDEKAR and MILDRED K. GORDON

Cortical granules are found in the unfertilized eggs of many invertebrates and vertebrates, including mammals, but are essentially absent in fertilized eggs (Austin, 1968). The extruded contents of the granules are thought to play a rôle in the prevention of polyspermy (Austin, 1961). In the sea urchin, the cortical granule breakdown occurs after sperm penetration has begun and appears to be a propagated reaction over the egg surface (Austin, 1961). Szollosi (1967), studying cortical granules in the hamster and rat, found that the granule breakdown in these species is generally activated by the attachment of a spermatozoon to an egg. Pikó (1969) reported that the reaction is triggered when membrane fusion and breakdown of the postnuclear cap region of the spermatozoon has begun. Thus, the presence

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Cecilia L. Schmidt, June Z. Kendall, Pramila V. Dandekar, M. M. Quigley and Karmen L. Schmidt

Summary. To determine the effects of prolonged hCG treatment in vitro upon granulosa cells from follicles of various sizes previously exposed to clomiphene citrate and hCG in vivo, progesterone and relaxin concentrations of spent media were correlated with light microscopic and ultrastructural characteristics. Intact, freshly dispersed cells were characterized by numerous lipid droplets, elliptical mitochondria with tubular or lamellar cristae, moderate rough-surfaced endoplasmic reticulum (RER), sparse smooth-surfaced endoplasmic reticulum (SER), and few Golgi. After 10–24 days in culture, 2 morphologically distinct cell types, 'granulosa-type' and 'luteal-type', were noted at the light microscopic level. Ultrastructurally, lipid droplets decreased in number, mitochondria became pleomorphic, RER became more prominent and dilated, and Golgi became more widely dispersed. Tubular SER became abundant and annular nexuses became more numerous after hCG treatment in vitro. Granulosa cells generated from all follicles responded to hCG treatment with significantly increased progesterone secretion after 4 days in culture. Relaxin was not detectable in any sample of medium. This study shows that human granulosa cells from 15–25-mm follicles retain their differentiated function of progesterone secretion in long-term culture and recover responsiveness to hCG in vitro, as demonstrated by enhanced progesterone secretion and development of prominent SER and increased annular nexuses.