Effect of maternal fatness on fetal steroids and semi-quantitative real-time PCR expression of receptor genes in sheep.

Sexual differentiation of the brain occurs between d 30 and 70 in the fetal lamb. The objective of this experiment was to determine if maternal fatness affects fetal steroid production and expression of their receptors which may ultimately alter endocrine systems postnatally. Fetuses were collected from ewes fed at either 100% (Control; n=5) or 150% (Fat; n=6) of NRC recommendations from 60 d prior to breeding until collection at 75 d of gestation. Hypothalamic and amygdala neural tissues were collected from twin male/female fetuses. Serum concentrations of testosterone were greater (P<0.001) in male fetuses compared to female fetuses. Further, male fetuses from Fat ewes had greater (P<0.05) serum concentrations of testosterone than male fetuses from Control ewes, but differences in testicular steroidogenic enzyme mRNA were not detected (P=0.18). Quantity of hypothalamic mRNA for estrogen receptor (ER) beta tended (P=0.1) to be influenced by a sex by treatment interaction. Messenger RNA for ER-beta was greater in female fetuses than male fetuses from Control ewes (P=0.05). Although amount of ER-beta mRNA did not differ among male fetuses (P=0.7), amounts tended to be less (P=0.07) in female fetuses from Fat ewes compared to those from Control ewes, and did not differ (P> or =0.8) from male fetuses. Hypothalamic ER-alpha mRNA tended (P=0.1) to be less in fetuses from Fat ewes compared to Control fetuses but was not influenced (P=0.3) by fetal sex or their interaction. Amount of mRNA for hypothalamic progesterone receptor tended (P=0.06) to be greater in male fetuses than female fetuses and tended to be less (P=0.06) in fetuses from Fat ewes than in Control fetuses, but did not differ by any sex by treatment interaction (P=0.6). Hypothalamic RNA for the androgen receptor did not differ by sex, dam nutritional treatment, or the interaction. Likewise, amygdala RNA for the estrogen or androgen receptor did not differ (P> or =0.3) by sex, treatment, or their interaction. Dam fatness appears to decrease the expression of progesterone receptor, ER-alpha, and decrease amount of ER-beta in the female fetuses while increasing circulating concentrations of testosterone in male fetuses. Altered expression of hypothalamic receptor genes by the uterine environment may affect adult responses to stress, sexual behavior and/or the pattern of gonadotropin release in response to gonadal steroids.

Localization of ISG15 and conjugated proteins in bovine endometrium using immunohistochemistry and electron microscopy.

The interferon-stimulated gene ISG15, a ubiquitin homolog, becomes conjugated to and regulates uterine proteins in response to conceptus-derived interferon-tau on d 18 of pregnancy. It was hypothesized here that cellular localization of ISG15 within endometrial cells might provide insight regarding function. Uteri were collected from cows (approximately 21-d estrous cycle) on d 17-21/0 of the estrous cycle and pregnancy and d 23, 45, and 50 of pregnancy. Intracellular ISG15 and its conjugates were present on d 17 of pregnancy, peaked to highest levels from d 18 to 23 and then declined to low but detectable levels by d 45 (P < 0.05) based on Western blotting. ISG15 and its conjugates were not detected on d 50 of pregnancy or during the estrous cycle. Immunohistochemistry revealed that ISG15 was localized throughout the endometrium on d 18-23, with heaviest staining in the sublumenal stratum compactum and the glandular epithelium throughout the stratum spongiosum. By d 45 and 50, ISG15 was lightly stained only in the stratum compactum immediately beneath the lumenal epithelium. Using transmission electron microscopy and immunogold labeling, ISG15 was specifically localized to organelles and compartments of endometrial epithelial cells and stromal cells: nucleus, perinuclear space, cytosol, mitochondria, rough endoplasmic reticulum, and cell membrane. This specific localization in epithelial and stromal cells led to the conclusion that ISG15 has diverse intracellular functions. The sustained presence of conjugated ISG15 through d 50 of pregnancy might reflect stabilization of conjugated proteins in response to implantation and the development of the placenta.