Involvement of molecules related to angiogenesis, proteolysis and apoptosis in implantation in rhesus monkey and mouse.

We have established the well-defined cycling, pseudo-pregnant and pregnant rhesus monkey models, and used these to analyze expression of the common molecules specifically related to angiogenesis, apoptosis or proteolysis, such as vascular endothelial growth factor (VEGF) and its receptors KDR, flt-1, flt-4 and flk-1, basic fibroblast growth factor (bFGF) and its receptors Flg, transforming growth factor-alpha and beta1 (TGF-a/beta1), and TGF-beta1 receptor type I (TbetaR-I) and type II (TbetaR-II), as well as steroidogenic acute regulatory protein (StAR), tissue type plasminogen activator/urokinase plasminogen activator/plasminogen activator inhibitor type 1 (tPA/uPA/PAI-1) and matrix matalloproteinase type 1, -3/tissue inhibitor matalloproteinase type 1, -2, -3 (MMP-1, -3/TIMP-1, -2, -3), Fas/FasL, BcL-2/Bax, in the corpus luteum (CL), in the functional layer of the endometrium and in the materno-fetal boundary of the implantation site. We have demonstrated that: expression of these molecules in the monkey CL, endometrium and materno-fetal boundary of the implantation site is correlated well with CL functional and vascular development and with the processes involved in the establishment of the implantation window as well as with the early stages of placentation. A coordinated increase in tPA and its inhibitor PAI-1 expression in the monkey and rat CL may be instrumental in initiating luteal regression in both species, and correlated well with the timing of the closure of the implantation window, whereas high uPA activity in the CL is important for the early formation of the CL and for maintaining its function which is closely correlated to the period of establishment of the implantation window. Apoptosis, proteolysis and angiogenesis occur in the CL and in the endometrium during the time of establishment of the implantation window, as well as in the materno-fetal boundary of the implantation site at the early stages of placentation. It seems that these processes occur in these tissues in a coordinated and time- and cell-dependent manner, and are reliant on each other. Based on these observations, we have designed experiments to test the actions of some related available compounds on mouse implantation, used alone or in combination. The preliminary data showed that the compounds which could effectively affect apoptosis, angiogenesis or proteolysis in the implantation site were capable of effectively inhibiting implantation by acting on the endometrium and/or on the CL. Furthermore, the combined use of these compounds produced an obvious additive effect on inhibiting implantation. This finding suggested this may be a good approach for developing an anti-implantation agent.

Disrupted expression of intermediate filaments in the testis of rhesus monkey after experimental cryptorchidism.

Cytoskeletons in Sertoli cell play an important role in process of spermatogenesis. The expression and distribution of the intermediate filaments, vimentin, keratin and desmin, were studied in the Sertoli cells of the cryptorchid testis of rhesus monkey. Vimentin was localized in the perinuclear region of Sertoli cells of the normal testis. An intense increase in vimentin immunoreactivity was observed with appearance of disorganized staining in the Sertoli cells of the cryptorchid testes. Cytokeratin 18, a marker of immature Sertoli cells, re-expressed in the cells of the adult cryptorchid testes. Desmin was also observed in the Sertoli cells in addition to the peritubular myoid cells on 30 days after the cryptorchid operation. These data suggest that Sertoli cells in primate can be affected by the heat stress. The altered changes in intermediate filaments could be possible to induce the Sertoli cell functional changes that would partially contribute to the germ cell apoptosis leading to azoospermia or oligozoospermia.

Tissue-type plasminogen activator and its inhibitor plasminogen activator inhibitor type 1 are coordinately expressed during ovulation in the rhesus monkey.

Ovulation is a gonadotropin-controlled process that is essential for the propagation of all mammalian species. In the present study, we used a pregnant mare serum gonodotropin/human chorionic gonadotropin (hCG)-induced, synchronized ovulation model in rhesus monkeys and systematically investigated the roles of the plasminogen activator (PA) system in the ovulatory process of the primate. At different follicular developmental stages throughout the periovulatory period, samples of ovaries, granulosa cells, and theca-interstitial cells as well as follicular fluid were collected, and levels of PA and PA inhibitor type-1 (PAI-1) were evaluated by fibrin overlay, reverse fibrin overlay, Northern blot analysis, and in situ hybridization, respectively. We showed that in response to an injection of ovulation-triggering hCG, which mimics the preovulatory surge of LH in the circulation, granulosa cell-derived tissue-type PA (tPA) was substantially elevated in preovulatory follicles and reached its maximum level just before ovulation. Although theca-interstitial cell-derived PAI-1 was also stimulated by pregnant mare serum gonodotropin and hCG treatments, however, the maximum level of PAI-1 appeared 12 h earlier than that of tPA. When ovulation approached, accompanying the highest tPA level in the preovulatory follicles, the follicular PAI-1 level declined dramatically to its minimum value. Moreover, our data on the expression of follicular PA and PAI-1 over the periovulatory period were reinforced by results obtained at the mRNA level. Our data suggest that the coordinated expression of tPA and PAI-1 may be of importance for the follicular rupture process during ovulation in the primate.

Bcl-2 and Bax are involved in experimental cryptorchidism-induced testicular germ cell apoptosis in rhesus monkey.

Apoptosis occurs spontaneously during spermatogenesis. However, little is known about its regulation in primate. Using an experimental cryptorchidism model in rhesus monkey, we have investigated the relationship between apoptosis and the Bcl-2 family members, Bcl-2 and Bax. Apoptotic cells were identified by in situ end labeling of fragmented DNA. The expressions of Bcl-2 and Bax in the testes during the heat stress-induced testicular germ cell apoptosis were detected by immunohistochemistry and Western blot techniques. The results showed that the apoptotic signals increased after heat treatment and the most susceptible cell types were spermatocytes and spermatids. A redistribution of Bax from the cytoplasmic to nuclear localization in some germ cells was observed. However, its total expression levels in the cells remained unchanged in the cryptorchid testes as determined by Western blot analysis; on the other hand, Bcl-2 levels increased significantly in response to heat stress. The subcellular redistribution of Bax and the increase in Bcl-2 expression in the cryptorchid testis suggest an involvement of Bcl-2 family members in heat stress-induced germ-cell apoptosis.

Expression and regulation of plasminogen activators, plasminogen activator inhibitor type-1, and steroidogenic acute regulatory protein in the rhesus monkey corpus luteum.

The corpus luteum (CL) is a transient endocrine organ that secretes progesterone to support early pregnancy. Using primate materials obtained from rhesus monkeys, we have in this study investigated the expression and regulation of the plasminogen activators (PAs) and PA inhibitor type 1 (PAI-1) during CL development and regression. Adult (5-7 yr old) female rhesus monkeys were treated with pregnant mare serum gonadotropin/human chorionic gonadotropin to induce ovulation and follicular luteinization. At various luteal developmental stages, CL or whole ovaries were obtained for preparing luteal cells, Northern blot, in situ hybridization, and immunohistochemistry. We demonstrated that luteal cells from the rhesus monkey were able to produce both tissue type PA (tPA) and urokinase type PA, as well as the physiological PAI-1. During luteal development in the monkey, urokinase type PA was the major PA species taking part in the active angiogenesis and tissue remodeling processes in the forming CL. However, the mRNA as well as the enzymatic activity levels of tPA increased dramatically in monkey CL with the advent of luteolysis. This change of tPA levels was in a temporal coordination with the regulation of PAI-1 expression, resulting in an increased tPA activity at the initiation of luteolysis. Therefore, we suggest that tPA might be a luteolytic factor to the monkey CL. A PAI-1 modulated tPA activity might be important for the initiation of luteolysis in the monkey. In addition, we have also demonstrated that the expression of steroidogenic acute regulatory protein in the monkey CL was in accordance with the changes of progesterone production, suggesting that steroidogenic acute regulatory protein expression may be considered as a reliable marker for CL function in primates.

Expression of Hsp70-2 in rhesus monkey testis during germ cell apoptosis induced by testosterone undecanoate.

Hsp70-2 functions as a molecular chaperone that assists other proteins in their folding, transport and assembly into complexes, and is postulated to be linked to the mechanisms that inhibit apoptosis. Here we have determined the association between Hsp70-2 gene and germ cell apoptosis induced by a high dose of testosterone undecanoate (TU). In this study, in situ analysis of cell DNA fragmentation and expression of Hsp70-2 in TU-treated monkey testes were compared with the normal testes. The TUNEL analysis data showed that a large number of germ cell apoptosis occurred in the testes on Day 30 after TU injection. Therefore, we speculate that spermatogenesis failure in TU-treated monkey testis may be a result of the germ cell apoptosis induced by a high dose of TU. As compared with that of normal testes, however, the level of Hsp70-2 mRNA was only slightly decreased while that of Hsp70-2 protein was almost unchanged in the testes from Day 7 to day 30 at the early stage of the germ cell apoptosis after TU treatment, but the levels of both Hsp70-2 mRNA and protein dropped dramatically on Day 60 when a large number of germ cells had undergone apoptosis and were depleted. Therefore, it is suggested that the Hsp70-2 may be not a molecule to prevent germ cell apoptosis induced by injection of TU in the testes at the early stage.