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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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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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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.