Regulation of Prohibitin Expression During Follicular Development: DISCUSSION(7)

DISCUSSION(7)

Sequestration of prohibitin in the nuclear compartment of aberrant embryos is compatible with the proposed role of prohibitin in transcriptional control.

At this stage, data to ascribe an antiproliferative or cell survival role to prohibitin in the ovaries are inconclusive. However, these results suggest a potential multifunctional role for prohibitin in the regulation of follicle cell growth and differentiation during follicular maturation and atresia. The changes in prohibitin expression and localization may be reflective of its antiproliferative activities, although the functional implications of these observations are currently unknown.

Regulation of Prohibitin Expression During Follicular Development: DISCUSSION(6)

Nonetheless, caution should be exercised in assessing the potential role and regulation of ovarian prohi-bitin expression, because measuring total prohibitin content may have limited physiologic significance. Potentially, the acidic isoform of the protein could well be the active form of prohibitin. Currently, we are investigating the site of phosphorylation of prohibitin using high-performance liquid chromatography/mass spectrometry techniques. The accumulation of prohibitin was observed in the aberrant embryos after NT and IVF followed by heat shock.

Regulation of Prohibitin Expression During Follicular Development: DISCUSSION(5)

DISCUSSION(5)

Whether this regulatory mechanism exists in the antiapoptotic action for prohibitin in the ovary remains to be determined. We observed translocation of prohibitin from the cytoplasm to the nucleus in granulosa cells of atretic follicles, although the significance of this observation is not immediately apparent. The mechanism by which prohi-bitin interacts with established cell death pathways to regulate apoptosis is also not known.

Regulation of Prohibitin Expression During Follicular Development: DISCUSSION(4)

Apoptosis in rat atretic follicles is confined primarily to granulosa cells. Irrespective of the stage of follicular maturation, theca cells in all atretic follicles were TUNEL-nega-tive. Interestingly, prohibitin was highly expressed in theca cells, and its immunoreactivity progressively increased with follicular maturation. The expression of prohibitin in theca cells was much higher than that in granulosa cells. Although speculative, increased expression of prohibitin at this stage likely may be preventing theca cells from undergoing apo-ptosis.

Regulation of Prohibitin Expression During Follicular Development: DISCUSSION(3)

DISCUSSION(3)

As the follicles matured toward the preovulatory stage, increased prohibitin expression was observed in the theca-interstitial cells, which also had increased P450scc enzyme activity. These findings are in agreement with those of previous studies concerning the expression of this steroidogenic factor during follicular development in the rat ovary.

Regulation of Prohibitin Expression During Follicular Development: DISCUSSION(2)

Immunohistochemical analyses revealed that prohibitin was highly expressed in the cytoplasm and perinuclear region of granulosa cells of some, but not all, preantral follicles. This pattern of prohibitin immunostaining appeared to be inversely related to that of PCNA expression and remained consistent during subsequent stages of follicular growth. This apparent heterogeneous expression pattern of prohibitin may be indicative of a diverse population of growing cells.

Regulation of Prohibitin Expression During Follicular Development: DISCUSSION(1)

 DISCUSSION(1)

Utilizing well-characterized models for gonadotropin stimulation, withdrawal in the rat ovary, and porcine oogenesis, we report here, to our knowledge for the first time, the cellular localization and modulation of prohibitin expression during rat follicular maturation, atresia, and porcine oocyte/embryo development.

Regulation of Prohibitin Expression During Follicular Development: RESULTS(6)

Localization of Prohibitin in Isolated Germinal Vesicle Oocytes and In Vitro Fertilized and Cloned Embryos

The rat and mouse prohibitin genes are functional ho-mologues of human prohibitin. This gene is highly conserved, because comparative analysis of the amino acid sequences indicates a single amino acid substitution between these three species. We have determined that the protein expression patterns of prohibitin in rat ovarian follicles are similar to that observed in porcine follicles (data not shown).

Regulation of Prohibitin Expression During Follicular Development: RESULTS(5)

RESULTS(5)

Interestingly, not all granulosa cells from early antral follicles exhibited immunostaining for pro-hibitin. As the follicles matured to the large antral follicle stage, granulosa cells in the follicular wall, but not those surrounding the oocyte, expressed P450scc (Fig. 3F). A gradient-like pattern of prohibitin expression was also observed in the large antral follicle (Fig. 3E).

Regulation of Prohibitin Expression During Follicular Development: RESULTS(4)

To determine whether prohibitin expression patterns correlated with the differentiated status of the cells, prohibitin and P450scc were immunolocalized in ovarian tissue sections from rats treated with eCG plus NRS. As shown in Figure 3A (see Table 1), prohibitin immunostaining was predominantly confined to the granulosa cells, whereas the theca-interstitial cells showed low staining patterns within the preantral follicles. In contrast, immunofluorescence labeling for P450scc was confined to the belt-like regions of the interstitial cells (Fig. 3B). The theca cells showed low immunoreactivity in the preantral follicles for this protein (Fig. 3A), whereas the granulosa cells were clearly devoid of P450scc (Fig. 3B).