Deletion of RhoA in Progesterone Receptor–Expressing Cells Leads to Luteal Insufficiency and Infertility in Female Mice

Abstract

Ras homolog gene family, member A (RhoA) is widely expressed throughout the female reproductive system. To assess its role in progesterone receptor-expressing cells, we generated RhoA conditional knockout mice RhoAd/d (RhoAf/f-Pgr-Cre+/−). RhoAd/d female mice had comparable mating activity, serum luteinizing hormone, prolactin, and estradiol levels and ovulation with control but were infertile with progesterone insufficiency, indicating impaired steroidogenesis in RhoAd/d corpus luteum (CL). RhoA was highly expressed in wild-type luteal cells and conditionally deleted in RhoAd/d CL. Gestation day 3.5 (D3.5) RhoAd/d ovaries had reduced numbers of CL, less defined corpus luteal cord formation, and disorganized CL collagen IV staining. RhoAd/d CL had lipid droplet and free cholesterol accumulation, indicating the availability of cholesterol for steroidogenesis, but disorganized β-actin and vimentin staining, indicating disrupted cytoskeleton integrity. Cytoskeleton is important for cytoplasmic cholesterol movement to mitochondria and for regulating mitochondria. Dramatically reduced expression of mitochondrial markers heat shock protein 60 (HSP60), voltage-dependent anion channel, and StAR was detected in RhoAd/d CL. StAR carries out the rate-limiting step of steroidogenesis. StAR messenger RNA expression was reduced in RU486-treated D3.5 wild-type CL and tended to be induced in progesterone-treated D3.5 RhoAd/d CL, with parallel changes of HSP60 expression. These data demonstrated the in vivo function of RhoA in CL luteal cell cytoskeleton integrity, cholesterol transport, StAR expression, and progesterone synthesis, and a positive feedback on StAR expression in CL by progesterone signaling. These findings provide insights into mechanisms of progesterone insufficiency.

Dietary exposure to mycotoxin zearalenone (ZEA) during post-implantation adversely affects placental development in mice

“…Therefore, food and water consumptions were rough estimations without considering gestation days. Body weights were recorded to determine pregnancy status as we reported previously [35].…”

“…Every 10 th cross placental sections in the largest middle part were collected. Haemotoxylin and Eosin staining (H&E) staining was done as previously described [35,36]. The middle three sections with the largest areas were selected for area quantification.…”

“…The middle three sections with the largest areas were selected for area quantification. The total area of each placental section and the areas of labyrinth layer, junctional zone, and decidua, which were outlined manually and quantified using Image J (National Institutes of Health, Bethesda, MD, USA) [35][36][37][38]. The average of each area from three sections in the same placenta was counted as one data point for statistical analysis.…”

Seipin deficiency leads to defective parturition in mice†

“…Uterine histology was performed as previously described [20,21]. Uterine area and myometrial area in uterine cross sections were measured using ImageJ [11,12,22].…”

“…Quantification of LC3 staining in D18.5 and postpartum day 1 (PPD1) uterine luminal epithelium (LE) was done as follow: LC3 staining in at least three representative LE areas and nearby myometrium areas (same size) from the same section was quantified using ImageJ [11,12,22]. The ratio of average LE staining intensity/average myometrium staining from one section per mouse was counted as one data point.…”