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Gonadotropin-releasing hormone (GnRH) and its agonist analog (GnRHa) are well known to have luteolytic effects. We previously reported that prolactin (PRL) stimulated matrix metalloproteinase (MMP)-2 activity to degrade collagen type IV as a mechanism of structural luteolysis. The effects of GnRHa treatment on developed corpora lutea are unknown. In this study we assessed the effect of GnRH on MMP expression and induction of structural involution of developed corpora lutea of superovulated rats using GnRHa. Pregnant mare serum gonadotropin-human chorionic gonadotropin (hCG)-synchronized ovulation and luteinization were induced in immature female rats, followed by daily treatment with GnRHa from 5 days after hCG treatment. GnRHa-induced involution of corpora lutea was evident 3 days after the treatment, as shown by their markedly smaller size (60% of the control weight). Nine days after hCG injection, serum progesterone and 20alpha-dihydroprogesterone concentrations were as low as those associated with structural luteolysis. These findings revealed that GnRHa has the ability to induce structural luteolysis in superovulated rats in the same way that PRL does. To gain information on mechanisms of luteal involution induced by GnRHa, we performed gelatin zymography. This showed a significant increase in the active form of MMP-2 in the luteal extract of GnRHa-treated rats (more than twofold that of the control). Activation of pro-MMP-2 by membrane type-MMP (MT-MMP) is reported to be a rate-limiting step for catalytic function. Another function of MT-MMP is to degrade collagen types I and III. The plasma membrane fraction of corpora lutea of GnRHa-treated rats activated pro-MMP-2 of fetal calf serum, resulting in a marked shift of the 68-kDa band to the 62-kDa band in the zymogram. A Northern hybridization study also revealed simultaneous significant increases in expression of MMP-2 mRNA and MT1-MMP mRNA in corpora lutea of GnRHa-treated rats (more than threefold the control level). In summary, hormonal and histological features of corpora lutea of GnRHa-treated superovulated rats correspond to those of structural luteolysis. GnRHa stimulated the expression of MMP-2 and MT1-MMP in developed corpora lutea associated with involution. These findings support the conclusion that MMP-2, activated by MT1-MMP, and MT1-MMP itself, remodel the extracellular matrix during structural luteolysis induced by GnRHa.

In general, growth hormone acts as a factor promoting cell proliferation in the positive direction and suppresses apoptosis. No report has described growth hormone (GH)-induced structural luteolysis. The present studies showed that GH induced structural luteolysis in rats after the induction of functional luteolysis by treatment with bromocriptine, and that apoptotic cells were present among luteal cells during structural luteolysis as shown by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. Zymography showed that the activity of matrix metalloproteinase (MMP)-2 increased during GH-induced structural luteolysis. The expression of c-myc protein of luteal cells was significantly decreased, but proliferating cell nuclear antigens (PCNA) were conversely increased during structural luteolysis, as shown by Western blot analysis. We propose that an excessive increase in PCNA and a marked decrease in c-myc protein of luteal cells lead to a disorder in the signals concerned with DNA synthesis, causing mitotic catastrophe and inducing apoptosis in luteal cells, and that structural luteolysis may be triggered. GH-induced apoptosis in structural luteolysis therefore highly depends on the cell cycle. There are thought to be two mechanisms of GH-induced structural luteolysis. One is apoptosis, and the other is destruction of extracellular matrix by MMP.

There are two stages of luteal regression. The first stage is functional regression that is characterized by a decreased production of progesterone secretion; the second stage of structural involution is referred to as a structural luteolysis. In rodents, prolactin has a biphasic action on the corpus luteum. It is luteotrophic, but when exposed to functionally regressed corpora lutea it causes luteolysis. The objective of the present studies was to examine mechanisms of prolactin action in structural luteolysis, whether apoptosis is involved in this process, and to examine the possible association of cell proliferation signals as mediators of structural luteolysis. Prolactin-induced structural luteolysis was associated with apoptosis verified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). Apoptotic cells made up about 3% of the cells 24 hours after the first injection of prolactin, a level that remained constant at all stages of structural luteolysis. Total ovarian weight and DNA content were decreased about 50% in 72 hours after induction of structural luteolysis by prolactin, The finding of about 3% of cells in apoptosis indicates apoptosis is a rapid process. Proliferating cell nuclear antigens (PCNA) of luteal cells were significantly decreased during functional luteal regression, but were conversely increased in structural luteolysis as shown by western blotting and immunohistochemistry. In general PCNA expression is reported to be decreased during structural involution, and there are no reports that have linked excess expression of PCNA with apoptosis and structural luteolysis. We speculate that an excessive increase in expression of PCNA which signals activation of cell proliferation creates a disorder in the signals involved with DNA synthesis. This disorder results in mitotic catastrophe and in the induction of apoptosis. Therefore the disorder of cell cycle signals in luteal cells are associated with prolactin induced apoptosis in structural luteolysis.

The human corpus luteum produces both estradiol and progesterone. It is well known that there are both autocrine and paracrine systems for the regulation of the corpus luteum and that estradiol regulates the progesterone production of the corpora lutea of some other species. To assess the direct effects of estrogen on human luteal function, we performed cell culture experiments. A low concentration of estradiol, almost equal to the amount of estradiol produced by human cultured luteal cells, directly stimulated progesterone production. 4-Cyclohexylaniline, an aromatase inhibitor, significantly reduced both progesterone production and estradiol production. Levels of estradiol higher than the levels that cultured human luteal cells themselves produced significantly reduced basal progesterone production and also significantly reduced human chorionic gonadotropin (hCG), forskolin and dibutyryl-cyclic AMP-stimulated progesterone production. According to these data, high doses of estradiol produced a luteolytic action which widely inhibited the steroidogenesis process. In conclusion, our results indicated that estradiol in part regulates progesterone production physiologically and blocks progesterone production in a pharmacological or pathological state in the human corpus luteum.