The variable expression of lectin-like oxidized low-density lipoprotein receptor (LOX-1) and signs of autophagy and apoptosis in freshly harvested human granulosa cells depend on gonadotropin dose, age, and body weight  Constanze Vilser, M.D., Heike Hueller, Ph.D., Marcin Nowicki, Ph.D., Fayez A. Hmeidan, M.D., Verona Blumenauer, Dipl.Biol., Katharina Spanel-Borowski, M.D., Ph.D.  Fertility and Sterility  Volume 93, Issue 8, Pages 2706-2715 (May 2010) DOI: 10.1016/j.fertnstert.2009.02.038 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 The study of lectin-like oxidized low-density lipoprotein receptor (LOX-1) in purified granulosa cells from seven different patient groups by using human recombinant LOX-1 protein as a positive control (+). Blots, left: Low stimulation with FSH (≤2,000 IU) appears to increase LOX-1 dimers (80 kDa) and oligomers independent of age and body mass index (BMI). Blots, right: A high FSH dose (>2,000 IU) apparently leads to low levels of LOX-1 dimers and oligomers in all four groups. Graph: The semiquantitative evaluation for the active LOX-1 forms (dimer and oligomer) reveals a similar trend. It becomes statistically significant for the group with an FSH dose ≤2,000 IU. Data are presented as mean ± SD. ∗P<.05; ∗∗P<.001. Micrograph: Numerous LOX-1–positive structures are aligned as point-like forms at the cell surface and in the perinuclear area. Immunostaining and confocal microscopy. Fertility and Sterility 2010 93, 2706-2715DOI: (10.1016/j.fertnstert.2009.02.038) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Light and transmission electron microscopy images of the granulosa cell morphology of different patient samples after purification of the follicle harvest from unwanted cells. Toluidine blue–stained semithin sections display (A) intact cells rich in lipid droplets as well as (B) light and dark cells with conspicuous vacuoles and a decrease in lipid droplets. Intact granulosa cells are shown with (C) abundant smooth endoplasmic reticulum and many lipid droplets, (D) typical autophagosomes as noted by the characteristic double membrane surrounding remnants of organelles (arrow), (E) defective mitochondria (arrow) with degenerating cristae and matrix, and (F) a multilamellar body. Forms of cell death are either (G) necrosis like with cyto- and chromatolysis or (H) reminiscent of apoptosis with peripheral DNA condensation and cell membrane blebbing. Fertility and Sterility 2010 93, 2706-2715DOI: (10.1016/j.fertnstert.2009.02.038) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 The study of purified granulosa cells for the autophagosome marker microtubule-associated light chain protein 3 (LC3) I by (A) Western blot analysis and (B) confocal microscopy. (A) Representative blots for LC3 I (cytosolic form) and LC3 II (lipidated membrane-bound form) for three different patient groups; the Neuro 2A cell line was used as a positive control (+).The two groups given high FSH doses (>2,000 IU) display strong LC3 II expression in the younger normal-weight patients (upper blot); older normal-weight women express both LC3 I and LC3 II forms (middle blot); in younger obese patients, both forms of LC3 are dramatically decreased under low FSH dose (lower blot). Graph: The semiquantitative evaluation for the LC3 II/LC I ratio from seven different patient groups reveals a higher ratio in the younger and normal-weight groups independent of the FSH dose. A higher LC3 II/LC I ratio is associated with a high FSH dose (>2,000 IU). Note that the ratio declines in an age– and body weight–dependent manner but independently of the hormone dose. Data are presented as mean ± SD. ∗P<.05; ∗∗P<.001. (B) The LC3 protein is associated with autophagosomes as indicated by ring-like structures (arrows) after immunostaining. Fertility and Sterility 2010 93, 2706-2715DOI: (10.1016/j.fertnstert.2009.02.038) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Western blot analysis of purified granulosa cells for (A) cleaved caspase-3 and (B) apoptosis-inducing factor (AIF). (A) The representative blots show no cleaved caspase-3 in the two groups; Jurkat cells treated with cytochrome c were used as a positive control (+) and untreated Jurkat cells as a negative control. For younger normal-weight patients receiving an FSH dose of ≤2,000 IU, mature caspase was noted (left). (B) Blots: The mitochondrial precursor of AIF (67 kDa) was strongly expressed; the 62-kDa AIF was the weak band in the two groups. The processed cytosolic form (mature AIF, 57 kDa) was strongly expressed in the older normal-weight group. The cortex of the aging brain was used as a positive control (+). Graph: The semiquantititative evaluation shows an increase in the 57-kDa AIF band in older normal-weight patients compared with younger obese patients under low FSH dose. Data are presented as mean ± SD. ∗P<.05; ∗∗P<.001. Fertility and Sterility 2010 93, 2706-2715DOI: (10.1016/j.fertnstert.2009.02.038) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 5 The percentage of dead granulosa cells in unpurified harvested follicles as determined by the trypan blue exclusion test. In the groups of younger normal-weight women, approximately 20% of the cells were dead, compared with 40%–50% in the other groups. The FSH dose had no effect. Data are presented as mean ± SD. ∗P<.05. Fertility and Sterility 2010 93, 2706-2715DOI: (10.1016/j.fertnstert.2009.02.038) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions