Characterization of long-term monolayer cultures of human granulosa cells from follicles of different size and exposed in vivo to clomiphene citrate and hCG

Abstract

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.

Ultrastructural characteristics of human granulosa cells in a coculture system for in vitro fertilization

“…5,7,and 8). The presence of a rich network of SER tubules and of a well-developed Golgi apparatus in the GCs confirm that luteinization is in progress (Amsterdam and Rotmensch, 1987;Bjersing, 1967;Schmidt et al, 1984;Suzuki et al, 1981). In this regard, isolated RER cisternae and ribosomes in luteinizing GCs may contribute to luteinization by synthesizing the enzymatic pattern involved in the steroid production (Crisp et al, 1970).…”

“…Although the loss of architectural and microtopographic integrity due to plating conditions can have marked effects on the structure and metabolism of cultured GCs (Zoller, 1991), fine structural changes superimposable to those described for GCs within the follicle occur spontaneously in vitro, after 12 h of culture in humans (Suzuki et al, 1981) or after 48-72 h in other mammals (Balboni and Zecchi, 1981;Zoller, 1991). These changes are further enhanced when LH/hCG, cyclic adenosine 3 0 5 0 -monophosphate (Soto et al, 1986), hCG-low density lipoproteins (Richardson et al, 1992), clomiphene citrate (Schmidt et al, 1984), or follicular fluid from large follicles (Zoller, 1991) are administered or if GCs are plated onto collagen- (Ben-Rafael et al, 1988) or matrigel-coated dishes (Hwang et al, 2000).…”

“…The junctional elements described may also represent the remnants of a welldeveloped junctional complex, formed by a tandem of gap and adherence junctions, that join neighboring GCs during differentiation inside the follicle (Amsterdam and Rotmensch, 1987;Rotmensch et al, 1986;Spanel-Borowski and Sterzik, 1987). Granulosa cell maturation and luteinization are in fact accompanied by a decrease in gap junctions and an increase in annular nexuses, the latter considered residual pre-existing linear gap junctions are internalized by endocytosis (Amsterdam and Rotmensch, 1987;Schmidt et al, 1984). Conversely, for other authors (Suzuki et al, 1981), annular gaps should be interpreted as actual (interdigitating) junctions.…”

“…Accumulation of lipids is one of the early signs of luteinization (Amsterdam and Aharoni, 1994;Crisp et al, 1970;Gulyas, 1984). Clusters of non-extractable, electrondense lipid droplets are numerous in GCs, both in vivo, especially in mural cells (Zoller, 1991) and in vitro, after culture (Schmidt et al, 1984). Lipid inclusions that increase in volume after the LH surge (or after LH/hCG administration in culture) contain precursors (cholesterol) for progesterone synthesis (Amsterdam and Rotmensch, 1987;Crisp et al, 1970;Dhar et al, 1996;Delforge et al, 1972;Gulyas, 1984;Van Voorhis, 1999).…”

Lipid droplets in cultured luteal cells in non-pregnant sheep fed different planes of nutrition

“…Lipid droplets in the CL or cultured luteal cells have been identified by using electron microscopy for several species including humans, rats, rabbits, cows and sheep (Blanchette, 1966;Enders, 1973;Schmidt et al, 1984;O'Shea et al, 1986O'Shea et al, , 1989Yuh et al, 1986;Fields et al, 1992;Fields and Fields, 1996;Towns et al, 1999). In our study, we not only identified presence and localization of LDs in LLC and SLC but we also quantified LDs demonstrating that the mean and median volume of individual LD was greater in SLC than LLC.…”