In vitro and in vivo effects of modulating TIMP1 on preimplantation embryo development and ovulation. In experiment 1, zygotes were collected from control rats and cultured for 24 h in the presence and absence of TIMP1. In experiment 2, Endo rats were treated with a TIMP1 function-blocking antibody or vehicle, and Sham rats were treated with TIMP1 or vehicle. Zygotes were examined for embryo quality, and ovaries were evaluated for anomalies in ovulation. In experiment 3, PF (peritoneal fluid) was collected from Endo and Sham rats from experiment 2. TIMP1 was immunoprecipitated out of a fraction of Endo PF. These three PF pools were injected into control rats. Ovaries were evaluated for ovulatory dysfunction.

Modulation of TIMP1 affects embryo development in vivo. The red color indicates proteasome labeling, which is expected to be concentrated in the pronuclei of normal zygotes and increased/aggregated in the cytoplasm of stressed cells. The green color indicates microtubule localization. Under control conditions, a pair of apposed male and female pronuclei, a landmark of normal fertilization and zygotic development, develop from the chromosomes of the oocyte metaphase-II plate and the fertilizing sperm nucleus, respectively. A, B) Zygotes collected from the vehicle-treated Sham rats. The microtubule network appeared normal (green). Apposed pronuclei are visible (arrowheads). In A, the blue color is DNA staining of the corona radiate cell nuclei. A′, B′) DIC images from the Sham zygotes. The arrows indicate the sperm tail. C, D) Zygotes collected from the vehicle-treated Endo rats. Pronuclei (arrowheads, blue) are abnormally small and failed to reach apposition. Proteasomes are aggregated throughout the cytoplasm, indicative of a cell under stress. Microtubule network is disarranged (green), indicative of the underdevelopment of these zygote. C′, D′) DIC images from the Endo zygotes. Arrows indicate sperm tail, which is not completely incorporated in the ooplasm in a zygote shown in D′. E) Zygote collected from an Endo rat treated with a TIMP1 function-blocking antibody. While the pronuclei (arrowheads, blue) are small and not yet apposed, an extensive, well-developed microtubule network can be seen (green). Proteasome staining is distributed diffusely in the cytoplasm, indicating a reduction of stress compared to nontreated rats. E′) DIC image of TIMP1 function blocking antibody-treated Endo rat. Arrow indicates the sperm tail. F) Although this female Sham rat treated with TIMP1 had spermatozoa present in the vaginal lavage, the oocytes failed to fertilize. Similar to the zygotes from the Endo rats, there is an increase in cytoplasmic proteasome labeling as well as clustering of microtubules in cytoplasmic foci/asters. Only a single set of (oocyte) chromosomes is present (arrowhead). F′) DIC image of TIMP1-treated Sham rat indicates abnormally shrunken cytoplasm; the sperm tail is not present. G) Unfertilized oocyte collected from a Sham rat treated with GM6001. This oocyte has a severely abnormal phenotype with proteasome-containing ring structures indicative of the process of cell stress or death. Arrowhead points to a hypercondensed chromatin, indicating an immature/defective oocyte. G′) DIC image of GM6001-treated Sham rat oocyte clearly showing the unique proteasome-containing ring structures induced by this treatment. H) Zygotecollected from a Sham rat treated with GM6001. The severely abnormal phenotype is repeated in this disintegrating zygote with proteasome-containing ring structures and corrupted plasma membrane. H′) DIC image of the same zygote, showing the familiar ring structures. Arrow indicates remnants of a sperm tail. Original magnification ×600.

Differential TIMP1 localization in the ovary after in vivo treatments. More TIMP1 protein was localized in the thecal cells of antral follicles in Endo rats (A) than in Sham rats (B). C) Treatment of Endo rats with a TIMP1 function-blocking antibody reduced TIMP1 localization to levels equal to Sham. D) Sham rats treated with TIMP1 had an increase in TIMP1 localization in the ovarian theca, providing further evidence that TIMP1 from the peritoneal cavity can sequester into the ovary to influence its function. The arrows indicate increased thecal localization of TIMP1 in A and D. Original magnification ×200.

Peritoneal fluid treatments affect TIMP1 protein localization in the ovary. Control rats treated with Endo PF (A) had more TIMP1 protein localization in thecal cells of antral follicles than control rats treated with TIMP1-depleted Endo PF (B) or Sham PF (C). Arrows point to the thecal layer. This provides further evidence that TIMP1 from the peritoneal cavity can be sequestered into the ovary, causing an imbalance between the MMPs and TIMPs. Original magnification ×200.

TIMP1 affects embryo development in vitro. A) Representative two-cell embryo cultured in control media for 24 h is morphologically normal with equal-sized blastomeres, equal-sized and -numbered nucleolus precursor bodies (arrowheads), and normal blastomere adhesion as visualized by DIC (differential interference contrast) microscopy. Remnants of the sperm flagellum (arrows), spanning both blastomeres, is still visible at this stage. Red indicates TIMP1 protein localization that is equally dispersed throughout the cytoplasm and nucleus. Green indicates NPC (nuclear pore complexes) that are concentrated around the nucleus (arrowheads), indicating good reformation of the nuclear envelope. Blastomere adhesion and the presence of NPC stacks in the cortical cytoplasm, probably in the form of annulate lamellae, increasing the bright green labeling area where adhering blastocysts contact each other. B) Representative two-cell embryo cultured with TIMP1 for 24 h is abnormal with elongated blastomeres, shrunken cytoplasm, and poor blastomere adhesion as well as abnormally small, supernumerary nucleoli (arrowheads in merged panel). Sperm flagellum, while split, is still prominent (arrow; DIC), suggesting a delayed postfertilization processing of sperm accessory structures. TIMP1 labeling is concentrated inside the nucleus and around the nucleoli (arrowheads in TIMP1 panel), possibly as a result of nuclear translocation of the endocytosed, extrinsic TIMP1 protein. NPC proteins are diffused across the cytoplasm, indicating dysfunctional nuclear envelope reassembly following embryo cleavage. Loose blastomere adhesion causes the lack of NPC signal between the blastomeres. Original magnification ×600.

Modulation of TIMP1 affects ovulation. Luteinized unruptured follicle syndrome was found in both Endo rats (A) and Sham rats treated with TIMP1 (B) but not in Sham rats (C). This is indicative of ovulatory dysfunction associated with excessive TIMP1. Corpora lutea (CL), follicles with granulosa cells (GC), and luteinized unruptured follicles (LUF) are noted. Original magnification ×200.

Modulation of TIMP1 affects ovarian TIMP1 inhibitory activity. A) A representative reverse zymogram. B) A representative Western blot analysis. C) Relative densitometry for TIMP1 inhibitory activity from reverse zymograms shows Endo rats and TIMP1-treated Sham rats had more TIMP1 inhibitory activity than Sham rats or TIMP antibody-treated Endo rats. Different lowercase letters indicate statistical significance (one-way ANOVA, P < 0.001). Error bars indicate ± SD.