Effects of immunization against bone morphogenetic protein-15 and growth differentiation factor-9 on ovarian function in mares.

Currently there is no contraceptive vaccine that can cause permanent sterility in mares. This study investigates the effect of vaccination against oocyte-specific growth factors, Bone Morphogenetic Protein 15 (BMP-15) and Growth Differentiation Factor 9 (GDF-9), on ovarian function of mares. It was hypothesized that immunization against these growth factors would prevent ovulation and/or accelerate depletion of the oocyte reserve. For this study, 30 mares were randomly assigned to three groups (n = 10/group) and vaccinated with BMP-15 or GDF-9 peptides conjugated to KLH and adjuvant, or a control of phosphate buffered saline and adjuvant. Horses received vaccinations at weeks 0, 6, 12, and 18. Ovarian activity and estrous behavior were evaluated 3 days a week via ultrasonography and interaction with a stallion. The study was initiated on March1, 2016. Upon evaluation of ovulation rate, the GDF-9 group did not have a difference (P = 0.66) in ovulation rate when compared to controls (10.8 and 10.0 ovulations, respectively), but the number of ovulations in the BMP-15 group was less (P = 0.02; 4.9 ovulations). Average follicle size prior to ovulation was less (P < 0.0001) in both treatment groups compared to controls. Estrous behavior was altered in both the BMP-15 and GDF-9 groups compared to controls after the second vaccination (P = 0.05 and 0.03, respectively). Although further research is required to determine the continued effects of vaccination against GDF-9 on ovulation rates, these results indicate that vaccination against BMP-15 and GDF-9 could serve as a contraceptive in wild horse populations.

Immune-mediated destruction of ovarian follicles associated with the presence of HSP90 antibodies.

We previously established that the presence of autoantibodies to heat-shock protein 90 (HSP90) is one common causes of female infertility, and demonstrated that its presence leads to detrimental effects on ovarian and reproductive function in mice. The pathophysiological mechanism and alteration in the immune physiology, however, remain unknown. We therefore carried out detailed analysis of various immune cells in the spleen and ovary following immunization of C57BL/6 female mice to generate antibodies to HSP90 in the general circulation. We observed a significant increase in levels of CD45- cells; CD4+ T cells; Ly6G6C+ cells; and CD11b+ Ly6G+ cells in the spleen of these mice, which correlate with the increased anti-HSP90 antibody production. Ovarian- and granulosa-cell populations also showed increased infiltration of CD45+ leukocytes and neutrophil and monocyte populations, which may have led to the observed ovarian follicular degeneration that predominantly manifested as empty follicles. A decrease in the number of functional ovarian follicles was also associated with a decrease in the level of Gdf9 gene expression. Thus, changes in the immune physiology of the spleen and ovary that leads to the generation of antibodies to HSP90 can also bring about the destruction of ovarian follicles.

Induced autoimmunity against gonadal proteins affects gonadal development in juvenile zebrafish.

A method to mitigate or possibly eliminate reproduction in farmed fish is highly demanded. The existing approaches have certain applicative limitations. So far, no immunization strategies affecting gonadal development in juvenile animals have been developed. We hypothesized that autoimmune mechanisms, occurring spontaneously in a number of diseases, could be induced by targeted immunization. We have asked whether the immunization against specific targets in a juvenile zebrafish gonad will produce an autoimmune response, and, consequently, disturbance in gonadal development. Gonadal soma-derived factor (Gsdf), growth differentiation factor (Gdf9), and lymphocyte antigen 75 (Cd205/Ly75), all essential for early gonad development, were targeted with 5 immunization tests. Zebrafish (n = 329) were injected at 6 weeks post fertilization, a booster injection was applied 15 days later, and fish were sampled at 30 days. We localized transcripts encoding targeted proteins by in situ hybridization, quantified expression of immune-, apoptosis-, and gonad-related genes with quantitative real-time PCR, and performed gonadal histology and whole-mount immunohistochemistry for Bcl2-interacting-killer (Bik) pro-apoptotic protein. The treatments resulted in an autoimmune reaction, gonad developmental retardation, intensive apoptosis, cell atresia, and disturbed transcript production. Testes were remarkably underdeveloped after anti-Gsdf treatments. Anti-Gdf9 treatments promoted apoptosis in testes and abnormal development of ovaries. Anti-Cd205 treatment stimulated a strong immune response in both sexes, resulting in oocyte atresia and strong apoptosis in supporting somatic cells. The effect of immunization was FSH-independent. Furthermore, immunization against germ cell proteins disturbed somatic supporting cell development. This is the first report to demonstrate that targeted autoimmunity can disturb gonadal development in a juvenile fish. It shows a straightforward potential to develop auto-immunization-based technologies to mitigate fish reproduction before they reach maturation. However, the highly variable results between treatments and individuals suggest significant optimization should be performed to achieve the full potential of this technology.

Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice.

The transforming growth factor ß (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

Effects of active immunization against growth differentiation factor 9 and/or bone morphogenetic protein 15 on ovarian function in cattle.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth in sheep, whereas only GDF9 is essential in mice suggesting that the roles of these oocyte-derived growth factors differ among species. At present, however, there is only limited information on the action of BMP15 and GDF9 in other species. Thus, the aim of this experiment was to determine the effect of neutralizing GDF9 and/or BMP15 in vivo on ovarian follicular development and ovulation rate in cattle through active immunization using the mature regions of the proteins or peptides from the N-terminal area of mature regions. Immunization with the BMP15 peptide, with or without GDF9 peptide, significantly altered (increased or decreased) ovulation rate. In some animals, there were no functional corpora lutea (CL), whereas in others up to four CL were observed. From morphometric examination of the ovaries, immunization with GDF9 and/or BMP15 reduced the level of ovarian follicular development as assessed by a reduced proportion of the ovarian section occupied by antral follicles. In addition, immunization against GDF9 and/or BMP15 peptides reduced follicular size to <25% of that in the controls. In conclusion, immunization against GDF9 and BMP15, alone or together, altered follicular development and ovulation rate in cattle. Thus, as has been observed in sheep, both GDF9 and BMP15 appear to be key regulators of normal follicular development and ovulation rate in cattle.