Novel Sperm and Gonadotropin-releasing Hormone-based Recombinant Fusion Protein: Achievement of 100% Contraceptive Efficacy by Co-immunization of Male and Female Mice.

Improvements in long-term female contraception can be achieved by vaccinating with sperm-derived proteins. Here, recombinant proteins comprising either (i) N- (amino acid residues 1-80) or C- (amino acid residues 76-126) terminal fragments of mouse sperm protein 17 (Sp17) fused to the promiscuous T non-B cell epitope of tetanus toxoid (TT), amino acid residues 830-844 followed by di-lysine linker (KK) (TT-KK-Sp17N or TT-KK-Sp17C , respectively) or (ii) mouse equatorin (amino acid residues 21-185) fused to the T non-B cell epitope of bovine RNase (amino acid residues 94-104) were expressed in Escherichia coli. Immunization of female FVB/J mice, using alum as an adjuvant, led to the generation of high antibody titers against the above proteins. Antibodies against both N- and C-terminal fragments of Sp17 reacted with the entire capacitated mouse spermatozoa, whereas those against equatorin reacted exclusively with the equatorial region. Despite the reactivity of all immune sera, only sera from mice immunized with TT-KK-Sp17N and TT-KK-Sp17C significantly reduced mouse in vitro fertilization. Mating studies of the immunized females with un-immunized male mice revealed the highest infertility in the TT-KK-Sp17C -immunized group. In an attempt to further boost the immune response, the C-terminal fragment of Sp17 was expressed as fusion protein with a tandem repeat of gonadotropin-releasing hormone (GnRH) (Sp17C -GnRH2 ). Immunization of both male and female mice with Sp17C -GnRH2 led to higher contraceptive efficacy compared to mice immunized with TT-KK-Sp17C . Interestingly, mating studies wherein partners were both immunized with Sp17C -GnRH2 showed a complete failure of female mice to conceive. Thus, immunization of both males and females with Sp17C -GnRH2 has the potential to increase contraceptive efficacy. Mol. Reprod. Dev. 83: 1048-1059, 2016. © 2016 Wiley Periodicals, Inc.

Constitutive expression of SMAR1 confers susceptibility to Mycobacterium tuberculosis infection in a transgenic mouse model.

BACKGROUND & OBJECTIVES: Studies involving animal models of experimental tuberculosis have elucidated the predominant role of cytokines secreted by T cells and macrophages to be an essential component of the immune response against Mycobacterium tuberculosis infection. The immune activities of CD4+ T cells are mediated in part by Th1 cytokine interferon gamma (IFN-γ) which is produced primarily by T cells and natural killer (NK) cells and critical for initiating the immune response against intracellular pathogen such as M. tuberculosis. Nuclear matrix protein SMAR1 plays an important role in V(D)J recombination, T helper cell differentiation and inflammatory diseases. In this study a transgenic mouse model was used to study the role of SMAR1 in M. tuberculosis infection. METHODS: Wild type BALB/c, C57BL/6, BALB/c-EGFP-SMAR1 and C57BL/6-SMAR1 transgenic mice were infected with M. tuberculosis (H37Rv). A dose of 100 bacilli was used for infection via respiratory route. Bacterial load in lung and spleen of infected mice was determined at 2, 4, 6 and 8 wk post-infection. Gene expression analysis for Th1 cytokines and inducible nitric oxide synthase (iNOS) was performed in infected lung tissues by quantitative reverse transcription (RT)-PCR. RESULTS: SMAR1 transgenic mice from both BALB/c and C57BL/6 genetic background displayed higher bacillary load and susceptibility to M. tuberculosis infection compared to wild type mice. This susceptibility was attributed due to compromised of Th1 response exhibited by transgenic mice. INTERPRETATION & CONCLUSIONS: SMAR1 transgenic mice exhibited susceptibility to M. tuberculosis infection in vivo irrespective of genetic background. This susceptibility was attributed to downregulation of Th1 response and its hallmark cytokine IFN-γ. Hence, SMAR1 plays an important role in modulating host immune response after M. tuberculosis infection.

Nucleosomal occupancy and CGG repeat expansion: a comparative analysis of triplet repeat region from mouse and human fragile X mental retardation gene 1.

The expansion of CGG repeats in the 5'-untranslated region (5'UTR) of FMR1 gene is the molecular basis of fragile X syndrome in most of the patients. The nature of the flanking sequences in addition to the length and interruption pattern of repeats is predicted to influence CGG repeat instability in the FMR1 gene. We investigated nucleosome occupancy as a contributor to CGG repeat instability in a transgenic mouse model containing unstable (CGG)(26,) from human FMR1 cloned downstream of nucleosome-excluding sequence. We observe that the transgene has an open chromatin structure compared to the stable endogenous mouse Fmr1 within the same nucleus. CGG repeats in mouse Fmr1 are flanked by nucleosomes unlike the repeats in the transgene in all the tissues examined. Further in vitro chromatin reconstitution experiments show that DNA fragment without the SV40ori/EPR (nucleosome-excluding sequence) forms more stable chromatin than the one containing it, despite having the same number of CGG repeats. The correlation between nucleosomal organisation of the FMR1 gene and CGG repeat instability was supported by significantly lower frequency of repeat expansion in mice containing an identical transgene without the SV40ori/EPR. Our studies demonstrate that flanking DNA sequences can influence repeat instability through modulation of nucleosome occupancy in the region.

Homeobox transcription factor Meis1 is crucial to Sertoli cell mediated regulation of male fertility.

BACKGROUND: Infertility has become a global phenomenon and constantly declining sperm count in males in modern world pose a major threat to procreation of humans. Male fertility is critically dependent on proper functioning of testicular Sertoli cells. Defective Sertoli cell proliferation and/or impaired functional maturation may be one of the underlying causes of idiopathic male infertility. Using high-throughput "omics" approach, we found binding sites for homeobox transcription factor MEIS1 on the promoters of several genes up-regulated in pubertal (mature) Sertoli cells, indicating that MEIS1 may be crucial for Sertoli cell-mediated regulation of spermatogenesis at and after puberty. OBJECTIVE: To decipher the role of transcription factor MEIS1 in Sertoli cell maturation and spermatogenesis. MATERIALS AND METHODS: Sc-specific Meis1 knockdown (KD) transgenic mice were generated using pronuclear microinjection. Morphometric and histological analysis of the testes from transgenic mice was performed to identify defects in spermatogenesis. Epididymal sperm count and litter size were analyzed to determine the effect of Meis1 knockdown on fertility. RESULTS: Sertoli cell (Sc)-specific Meis1 KD led to massive germ cell loss due to apoptosis and impaired spermatogenesis. Unlike normal pubertal Sc, the levels of SOX9 in pubertal Sc of Meis1 KD were significantly high, like immature Sc. A significant reduction in epididymal sperm count was observed in these mice. The mice were found to be infertile or sub-fertile (with reduced litter size), depending on the extent of Meis1 inhibition. DISCUSSION: The results of this study demonstrated for the first time, a role of Meis1 in Sc maturation and normal spermatogenic progression. Inhibition of Meis1 in Sc was associated with deregulated spermatogenesis and a consequent decline in fertility of the transgenic mice. CONCLUSIONS: Our results provided substantial evidence that suboptimal Meis1 expression in Sc may be one of the underlying causes of idiopathic infertility.

An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation.

Differential next-generation-omics approaches aid in the visualization of biological processes and pave the way for divulging important events and/or interactions leading to a functional output at cellular or systems level. To this end, we undertook an integrated Nextgen transcriptomics and proteomics approach to divulge differential gene expression of infant and pubertal rat Sertoli cells (Sc).Unlike, pubertal Sc, infant Sc are immature and fail to support spermatogenesis. We found exclusive association of 14 and 19 transcription factor binding sites to infantile and pubertal states of Sc, respectively, using differential transcriptomics-guided genome-wide computational analysis of relevant promoters employing 220 Positional Weight Matrices from the TRANSFAC database. Proteomic SWATH-MS analysis provided extensive quantification of nuclear and cytoplasmic protein fractions revealing 1,670 proteins differentially located between the nucleus and cytoplasm of infant Sc and 890 proteins differentially located within those of pubertal Sc. Based on our multi-omics approach, the transcription factor YY1 was identified as one of the lead candidates regulating differentiation of Sc.YY1 was found to have abundant binding sites on promoters of genes upregulated during puberty. To determine its significance, we generated transgenic rats with Sc specific knockdown of YY1 that led to compromised spermatogenesis.

An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation.

BACKGROUND: Spermatogonial stem cell (SSC) transplantation (SSCT) has become important for conservation of endangered species, transgenesis and for rejuvenating testes which have lost germ cells (Gc) due to gonadotoxic chemotherapy or radiotherapy during the prepubertal phase of life. Creating a germ cell-depleted animal model for transplantation of normal or gene-transfected SSC is a prerequisite for such experimental studies. Traditionally used intraperitoneal injections of busulfan to achieve this are associated with painful hematopoietic toxicity and affects the wellbeing of the animals. Use of testicular busulfan has been reported recently to avoid this but with a very low success rate of SSCT. Therefore, it is necessary to establish a more efficient method to achieve higher SSCT without any suffering or mortality of the animals. METHODS: A solution of busulfan, ranging from 25 µg/20 µl to 100 µg/20 µl in 50 % DMSO was used for this study. Each testis received two diagonally opposite injections of 10 µl each. Only DMSO was used as control. Germ cell depletion was checked every 15 days. GFP-expressing SSC from transgenic donor mice C57BL/6-Tg (UBC-GFP) 30Scha/J were transplanted into busulfan-treated testis. Two months after SSCT, mice were analyzed for presence of colonies of donor-derived SSC and their ability to generate offspring. RESULTS: A dose of 75 µg of busulfan resulted in reduction of testis size and depletion of the majority of Gc of testis in all mice within 15 days post injection without causing mortality or a cytotoxic effect in other organs. Two months after SSCT, colonies of donor-derived Gc-expressing GFP were observed in recipient testes. When cohabitated with females, donor-derived offspring were obtained. By our method, 71 % of transplanted males sired transgenic progeny as opposed to 5.5 % by previously described procedures. About 56 % of progeny born were transgenic by our method as opposed to 1.2 % by the previously reported methods. CONCLUSIONS: We have established an efficient method of generating a germ cell-depleted animal model by using a lower dose of busulfan, injected through two diagonally opposite sites in the testis, which allows efficient colonization of transplanted SSC resulting in a remarkably higher proportion of donor-derived offspring generation.

Evaluation of recombinant fusion protein comprising dog zona pellucida glycoprotein-3 and Izumo and individual fragments as immunogens for contraception.

Vaccines based on gamete specific proteins have been proposed for fertility inhibition. In the present study, immunogenicity and contraceptive potential of E. coli-expressed recombinant fusion protein TT-KK-ZP3-GGG-Iz, comprising promiscuous T cell epitope of tetanus toxoid (TT) followed by dilysin linker (KK), a fragment of dog zona pellucida glycoprotein-3 (ZP3), triglycine spacer (GGG) and a fragment of dog Izumo (Iz) without any affinity tag has been evaluated in female FvB/J mice. In addition, recombinant TT-KK-ZP3 and Izumo linked to the promiscuous T cell epitope of bovine RNase (bRNase-KK-Iz) and their physical mixture were also used. SDS-PAGE and immunoblot studies revealed ∼32kDa band corresponding to TT-KK-ZP3-GGG-Iz, ∼22kDa band of TT-KK-ZP3 and ∼11kDa band of bRNase-KK-Iz. Groups of mice immunized with the above recombinant proteins led to the generation of high antibody titres against the respective proteins. Immunization with recombinant TT-KK-ZP3-GGG-Iz generated higher antibody titre as compared to mice immunized with physical mixture of TT-KK-ZP3 and bRNase-KK-Iz. Antibodies against TT-KK-ZP3-GGG-Iz and TT-KK-ZP3 recognized mouse and dog ZP and those against TT-KK-ZP3-GGG-Iz and bRNase-KK-Iz recognized mouse and dog acrosome-reacted sperm in an indirect immunofluorescence assay. Immune sera from groups of mice immunized with the above recombinant proteins led to a significant reduction in mouse in vitro fertilization. Mating studies revealed significant reduction in fertility as compared to adjuvant control group. Highest infertility was observed in group of mice immunized with TT-KK-ZP3 followed by TT-KK-ZP3-GGG-Iz. Infertility was associated with the antibody titres against ZP3, whereas no association in the inhibition of fertility and antibody titres against Izumo was observed. In conclusion, these studies revealed the contraceptive potential of ZP3, which could not be further enhanced by the inclusion of Izumo.

Dickkopf homolog 3 (DKK3) plays a crucial role upstream of WNT/ß-CATENIN signaling for Sertoli cell mediated regulation of spermatogenesis.

Testicular Sertoli cells (Sc) are main somatic component of seminiferous tubules that govern the differentiation of germ cells (Gc) and provide them physical support. Sc are the target of follicle stimulating hormone (FSH) and testosterone (T) which are known to regulate spermatogenesis. FSH and T levels in human and sub-human male primates remain high during infancy (4-6 months post birth), similar to those during puberty. Subsequently, juvenile phase is marked with low levels of these hormones. In spite of prolonged hormonal exposure, spermatogenesis is not discerned during infancy unlike that during puberty. Situation during infancy is similar to certain idiopathic male infertility, where prolonged hormone supplementation fails to initiate spermatogenesis. In our quest to determine non hormonal causes of idiopathic infertility which may reside within the Sc, we investigated the association between spermatogenesis and Sc specific gene(s) expressed differentially during puberty and infancy. Although products of several genes may be necessary for quantitatively normal spermatogenesis, one needs to investigate their roles one by one. Differential display and real time PCR analysis revealed higher expression of a known tumor suppressor, Dickkopf homolog 3 (DKK3), by pubertal monkey Sc as compared to infant Sc. To evaluate role of DKK3 in spermatogenesis, we generated DKK3 knock down mice (DKDM) using shRNA construct targeted to DKK3. In testis of adult DKDM, expression of DKK3 mRNA and protein were significantly (p<0.05) low and was associated with elevated WNT-4/ß-CATENIN activity. Elevated ß-CATENIN activity is known to restrict Sc maturation. Abundant expression of infant Sc marker, Mullerian inhibiting substance (MIS), in the testes of adult DKDM confirmed lack of Sc maturation in DKDM. Gc differentiation and fertility was severely compromised in DKDM. This is the first report of role of DKK3 in the testis and DKK3 mediated regulation of spermatogenesis via WNT-4/ß-CATENIN modulation.

Immunogenicity and contraceptive efficacy of Escherichia coli-expressed recombinant porcine zona pellucida proteins.

PROBLEM: To overcome availability of the purified native zona pellucida (ZP) glycoproteins for immunocontraception, porcine ZP3, and ZP4 were expressed in E. coli. METHOD OF STUDY: Purified recombinant proteins were characterized by SDS-PAGE and Western blot, and immunogenicity and contraceptive efficacy determined in FvB/J female mice. RESULTS: Purified ZP3, ZP3 with promiscuous T-cell epitope of tetanus toxoid, ZP4 and ZP4 incorporating promiscuous T-cell epitope of bovine RNase revealed ~44-, ~49-, ~53-, and ~55-kDa bands by SDS-PAGE and Western blot, respectively. Immunization of female mice with recombinant proteins elicited high antibody titers as well as T-cell responses. Immune sera recognized mouse oocyte ZP and also inhibited in vitro fertilization. Immunized mice showed significant decrease in fertility. Recombinant proteins were able to recall memory antibody response in female mice primed with porcine native ZP. CONCLUSION: Availability of recombinant porcine proteins will be useful in the development of contraceptive vaccine.

Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning.

Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.