ABSTRACT
The present studies investigated the hypothesis that TGFbeta plays a role in mediating LH/hCG-induced maturation, ovulation and/or luteinization of follicles in the pig. In Experiment 1, the temporal and spatial gene expression patterns of TGFbeta signaling components were examined in pig follicles which had been induced to ovulate and luteinize in vivo by hCG treatment, or by the LH-surge. Pre-pubertal pigs were injected with PG-600 followed by hCG, and ovaries were collected surgically at 0, 1, 12, 24 and 48h post-hCG. Post-ovulatory follicles were also collected from cycling gilts on Day 4 (D4) of the estrous cycle. Pre- and post-ovulatory follicles were used for the measurement of mRNA (PCR) and protein (Western blots) abundance and for protein localization by immunohistochemistry (IHC). Steady state amounts of mRNA for TGFbeta3 and TGFbetaR2 were increased (P<0.05, as compared to 0h) at 12h and on D4, respectively, while TGFbeta2 protein showed a tendency to increase on D4. TGFbeta signaling components did not change significantly. By IHC, the localization of TGFbeta components was as follows: pre-ovulatory follicles; TGFbeta1 - granulosa cells (GC), TGFbeta2 - theca cells (TC), TGFbetaR1 and 2 - GC and TC: post-ovulatory follicles; TGFbeta1 and 2 and TGFbetaR1 and 2 - luteinizing TC and GC. In Experiment 2, TGFbeta1 (1-100ng/ml) alone had no significant effect on progesterone (P4) secretion by pig GC in culture. Furthermore, while LH+IGF-1 (positive control) stimulated P4 approximately 10-fold, TGFbeta at 10 and 100ng/ml added in combination with LH+IGF-1, had no effect on P4 accumulation. In conclusion, data from the present study on temporal and spatial patterns of expression of the TGFbeta-system suggest that TGFbeta may play a role in the overall process of luteinization, but it appears not to influence steroidogenesis in luteinizing pig follicles.
Subject(s)
Ovarian Follicle/metabolism , Ovulation/genetics , Ovulation/metabolism , Swine , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Animals , Aromatase/genetics , Aromatase/metabolism , Cells, Cultured , Female , Gene Expression Profiling , Ligands , Luteinization/blood , Luteinization/genetics , Luteinization/metabolism , Ovarian Follicle/physiology , Ovulation/blood , Phosphoproteins/genetics , Phosphoproteins/metabolism , Progesterone/blood , Progesterone/metabolism , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/genetics , Signal Transduction/physiology , Smad Proteins/genetics , Smad Proteins/metabolism , Swine/genetics , Swine/metabolism , Swine/physiology , Time Factors , Transforming Growth Factor beta/bloodABSTRACT
The estrogenic isoflavone genistein is a common dietary component that has been shown to affect reproductive development in experimental animals at high doses. The objective of the present study was to examine interactions of genistein and the hormonally active pesticide methoxychlor on mammary gland development in juvenile rats. Timed-pregnant Sprague-Dawley rats were fed a soy- and alfalfa-free diet containing different combinations of genistein (300 and 800 ppm) and methoxychlor (800 ppm). Rats were fed these diets starting on gestation day (GD)1 and continuing through pregnancy and lactation until postnatal day (PND) 22, when the pups were killed. Inguinal mammary glands from both female and male pups were processed as whole-mount preparations for morphometric analysis. The total glandular area and the numbers of branch points, lateral buds, and terminal end buds in the male rats were found to be significantly greater in the groups exposed to methoxychlor than those exposed to genistein only. These effects were not observed in the female rats. In the male rats, methoxychlor had the most prominent effect on elongating the glandular ducts, while genistein enhanced the ductile branching. The 2 compounds in combination promoted the development of alveolar-lobular structure, an effect not observed with either compound alone. Immunostaining for proliferating cell nuclear antigen revealed a high percentage of immunopositive cells in the mammary epithelia of the males exposed to methoxychlor and genistein (800 ppm) compared to the controls. While no significant changes in serum levels of mammotrophic hormones were detected, increased immunostaining for insulin-like growth factor-1 receptor, estrogen receptor alpha, and progesterone receptor in the genistein + methoxychlor group suggested that local factors involved in regulating mammary growth may have played a role in propagating the endocrine effects of these two compounds. These results indicated that the mammary glands of juvenile male rather than juvenile female rats may be more sensitive to certain endocrine-active compounds and that high levels of phytoestrogens have the potential to alter the toxicological behaviors of other hormone mimics.
