Progesterone Synthesis and Fine Structure of Dissociated Monkey (Macaca mulatta) Luteal Cells Maintained in Culture

Abstract

The process of functional regression of the primate corpus luteum in the menstrual cycle is not well understood, yet it remains an important determinant in fertility management, including contraceptive actions. Accordingly, the feasibility of maintaining rhesus monkey luteal cells in culture for long term studies was explored. When midluteal phase corpus luteum (CL) was dispersed with collagenase in Ham’s F10 (F10) medium, 60% of the initial 5 x 10⁴ cells plated attached. Unattached cells were removed in media changes on Day 2 and 4. Trypan blue exclusion showed that 60-80% of unattached cells were dead or dying. At 10 days of culture, the number of cells per dish in F10 was comparable to the number of cells when D-valine was substituted for L-valine (D-val-F10) and when 100 ng hCG/ml was added to F10. The number of cells was slightly higher when cultured in Hank’s M199 (M199) medium. Progesterone (P) production in F10 medium declined markedly between Day 2 and Day 6, but remained detectable until Day 10. Although hCG increased P production (P<0.01) throughout the 10 days of culture, it failed to prevent the pattern of diminished P production. Cells cultured in F10 for 6 days before exposure to hCG increased P production (P<0.01) during the next 2 days. P production in D-val-F10 was similar to F10 medium; however, in M199 medium it was 2-fold greater (P<0.01). Cytologically, a large and a smaller luteal cell population was identified after dissociation. The large cells had agranular ER (AER) distributed peripherally, whereas the smaller cells had more granular ER (GER) and Golgi. Except for the cultures containing hCG, cultured cells appeared similar. Regardless of composition of culture medium, 3-4% of the cells in culture were large. In these large cells, the zone of peripheral AER was absent and mitochondria became elongated with increased age in culture. The smaller luteal cells were the predominant type throughout the culture period. The smaller luteal cells in culture in F10, D-val-F10 and M199 had diminished quantities of GER and increased number of polysomes with advancing age in culture. Smaller luteal cells, maintained in the presence of hCG for 4 days of longer, had more AER, large ovoid nuclei and thread-like branching mitochondria. We present evidence that these monkey luteal cells can be maintained for at least 10 days in the condition described here.