Major Histocompatibility Complex-B haplotype and ovarian graft response.

Gonadal tissue transfer is considered one of the best methods to preserve genetic variability. Poultry hosts can receive a gonad from a donor of a different genetic background, sustain the growth of this graft, and produce gametes from it. Unfortunately, the host's strong immune response may significantly reduce the gonadal graft's ability to reach maturity. Our study aimed to evaluate the influence of MHC-B alleles in rejecting a gonadal graft of similar or different genetic backgrounds. In the first experiment, ovarian tissue was transplanted to chicks of similar genetic backgrounds, either Lohmann White (LW) with variable MHC-B or Barred Rock (BR) with fixed MHC-B. The sustained growth of donor ovarian tissues occurred in (4/7 hosts) BR (MHC-B matched) hosts only-one of these graft-positive-BR hens produced eggs derived from the donor ovary. No grafts were recovered when the host and the donor had an LW background (0/9; MHC-B mismatched). In the second experiment, ovarian transplantation was done between chicks of either similar or different genetic backgrounds (Brown Leghorn [BL], BR, and BL/BR F1). The 2 pure lines contained only one MHC-B allele, whereas the F1 heterozygotes had both. All host birds were given a daily dose of an immunosuppressant (mycophenolate mofetil) until maturity. The success rate was assessed by microsatellite genotype confirmation of donor-derived ovaries plus physiological and histological analyses of ovarian grafts. In this second experiment, 11 out of 43 ovarian hosts laid eggs. However, all fertilized eggs from these hens were derived from the remnant host ovarian tissue, not from the donor ovaries. A necropsy assessment was done on all 43 host birds. Ten donor grafts were recovered from hosts having matched (6 hosts) and mismatched (4 hosts) MHC-B, and none were functional. Interestingly, 6 of them were enclosed by a serous membrane capsule filled with fluid and had various tissue growth. In addition, clusters of immune cells were observed in all recovered donor grafts. Our results demonstrated that genetic background could greatly influence the success of gonadal transfer in chickens.

Cryopreservation of bison semen without exogenous protein in extender and its fertility potential in vitro and in vivo following fixed-time artificial insemination.

Successful cryopreservation of bison semen is fundamental for restoration of genetic diversity in Canada's wood bison. Conventional bovine semen extenders contain animal products, such as egg yolk and milk, which are undesirable because of biosecurity risks and undefined composition. In this study, we examined the efficacy of an exogenous protein-free extender containing cholesterol-cyclodextrin complex (CC) to cryopreserve bison semen. The study also provided an opportunity to determine the effectiveness of different ovulation synchronization protocols for fixed-time artificial insemination in bison. Semen was collected from wood bison bulls via electroejaculation and cryopreserved in either Tris-egg yolk-glycerol (called 'TEYG') extender or pretreated with CC (2 mg/mL semen) and diluted in Tris-glycerol (collectively called 'CC-TG') extender. Post-thaw sperm motion characteristics and in vitro fertilization of cattle oocytes confirmed that CC alone without egg yolk protected bison sperm during cryopreservation process. In the first fertility trial, however, no pregnancy was obtained following fixed-time artificial insemination of bison cows with CC-TG extender. In a follow-up trial, low concentration of CC (1 mg/mL semen) resulted in better post-thaw sperm motion characteristics, fertility rate, and birth of live calves following fixed-time artificial insemination. Results showed that 1 mg CC/mL semen completely replaced egg yolk in bison semen extender. In addition, both follicular ablation and steroid treatment protocols provided ovulation synchrony to permit successful application of fixed-time artificial insemination in bison. This is the first report on the birth of live bison calves following fixed-time artificial insemination using semen cryopreserved in a defined extender.

ENU-induced mutant allele of Dnah1, ferf1, causes abnormal sperm behavior and fertilization failure in mice.

Given attention to both contraception and treatment of infertility, there is a need to identify genes and sequence variants required for mammalian fertility. Recent unbiased mutagenesis strategies have expanded horizons of genetic control of reproduction. Here we show that male mice homozygous for the ethyl-nitroso-urea-induced ferf1 (fertilization failure 1) mutation are infertile, producing apparently normal sperm that does not fertilize oocytes in standard fertilization in vitro fertilization assays. The ferf1 mutation is a single-base change in the Dnah1 gene, encoding an axoneme-associated dynein heavy chain, and previously associated with male infertility in both mice and humans. This missense mutation causes a single-amino-acid change in the DNAH1 protein in ferf1 mutant mice that leads to abnormal sperm clumping, aberrant sperm motility, and the inability of sperm to penetrate the oocyte's zona pellucida; however, the ferf1 mutant sperm is competent to fertilize zona-free oocytes. Taken together, the various mutations affecting the DNAH1 protein in both mouse and human produce a diversity of phenotypes with both subtle and considerable differences. Thus, future identification of the interacting partners of DNAH1 might lead to understanding its unique function among the sperm dyneins.

Superovulation and embryo transfer in wood bison (Bison bison athabascae).

Two experiments were done to develop an effective superovulatory treatment protocol in wood bison for the purpose of embryo collection and transfer. In experiment 1, donor bison were assigned randomly to four treatment groups (N = 5 per group) to examine the effects of method of synchronization (follicular ablation vs. estradiol-progesterone treatment) and ovarian follicular superstimulation (single slow-release vs. split dose of FSH). Recipient bison were synchronized with donor bison by either follicular ablation (N = 8) or estradiol-progesterone treatment (N = 9). In experiment 2, bison were assigned randomly to four treatment groups (N = 5 per group) to examine the ovarian response to two versus four doses of FSH, and the effect of progesterone (ovarian superstimulation with or without an intravaginal progesterone-releasing device). Donor bison were inseminated with fresh chilled wood bison semen 12 and 24 hours after treatment with GnRH (experiment 1) or LH (experiment 2). The ovarian response was assessed using ultrasonography. In experiment 1, the number of large follicles (≥ 7 mm) increased in response to both FSH treatments, but the diameter of the largest follicle detected 4 and 5 days after the start of ovarian superstimulation was greater in bison treated with a single dose of FSH than in those treated with two doses (P < 0.05). A total of 10 ova and/or embryos were collected. One blastocyst was transferred to each of five recipient bison resulting in the birth of two live wood bison calves. In experiment 2, two doses of FSH resulted in a greater number of large follicles (≥ 9 mm) on Days 4, 5, and 6 (P < 0.05) after beginning of superstimulation (Day 0), and more ovulations than four doses of FSH (11.2 ± 2.4 vs. 6.4 ± 0.8; P < 0.05). Embryo collection was performed on only five donors, and a total of 19 ova and/or embryos were recovered. In summary, fewer FSH treatments were as good or better than multiple treatments, consistent with the notion that minimizing handling stress improves the superovulatory response in bison. Follicular ablation and estradiol plus progesterone treatment were effective for inducing ovarian synchronization in embryo donor and recipient bison, and an intravaginal progesterone-releasing device during superstimulatory treatment did not influence the superovulatory response or embryo collection. Delaying ovulation-inducing treatment (GnRH or LH) to 5 days after superstimulatory treatment resulted in a greater number of ovulations and improved embryo collection efficiency (experiment 2). Embryo collection and transfer resulted in live offspring from wild wood bison.

AKAP9 is essential for spermatogenesis and sertoli cell maturation in mice.

Mammalian male fertility relies on complex inter- and intracellular signaling during spermatogenesis. Here we describe three alleles of the widely expressed A-kinase anchoring protein 9 (Akap9) gene, all of which cause gametogenic failure and infertility in the absence of marked somatic phenotypes. Akap9 disruption does not affect spindle nucleation or progression of prophase I of meiosis but does inhibit maturation of Sertoli cells, which continue to express the immaturity markers anti-Mullerian hormone and thyroid hormone receptor alpha in adults and fail to express the maturation marker p27(Kip1). Furthermore, gap and tight junctions essential for blood-testis barrier (BTB) organization are disrupted. Connexin43 (Cx43) and zona occludens-1 are improperly localized in Akap9 mutant testes, and Cx43 fails to compartmentalize germ cells near the BTB. These results identify and support a novel reproductive tissue-specific role for Akap9 in the coordinated regulation of Sertoli cells in the testis.

Mutagenesis as an unbiased approach to identify novel contraceptive targets.

To accommodate diverse personal needs in family planning, diverse contraceptive approaches are desirable. This goal requires identification of new contraceptive targets. Phenotype-driven mutagenesis is an unbiased approach to identify novel genes and functions in reproductive processes. The ReproGenomics Program at The Jackson Laboratory is a United States National Institutes of Health resource for production, identification and distribution of mutant mouse models of infertility that can be used for identification of potential targets for contraception. The strategy of this program is whole genome, random ENU mutagenesis, coupled with a phenotype screen for breeding failure as the only phenotype. A three-generation breeding scheme selects recessive mutations affecting reproductive functions. G3 males and females that fail to reproduce by natural mating to wild-type animals undergo secondary phenotype screens to assess gonad and accessory organ histology, hormone production, gamete production and gamete function in fertilization. The genetic transmission of the infertility trait in each family is confirmed and each mutation is genetically mapped to a defined chromosome region, facilitating identification of candidate genes from sequence and expression databases. Genes essential for fertility in both males and females and acting both meiotically and post-meiotically have been identified by this strategy. Phenotypes include male infertility with normal sperm count, but failure in fertilization of oocytes. Phenotype descriptions of each mutation are posted on the program website, . These unique reproductive mutant mouse resources will lead to new discoveries in andrology (and gynecology) research, as well as reproductive medicine. Dissection of gene function in known and newly discovered reproductive pathways will expand our focus to reveal novel targets for contraception.

Polyol pathway along the bovine epididymis.

During the epididymal transit, male gametes acquire new surface proteins necessary for their fertilizing ability. We have previously shown that membranous vesicles, called epididymosomes, interact with sperm surface within the epididymal fluid allowing transfer of some proteins to different subcellular compartments of spermatozoa. We previously showed that one of the major proteins associated with epididymosomes was an aldose reductase (gene: AKR1B5) and confirmed that aldose reductase is located in the epithelial cells bordering the intraluminal compartment of the epididymis. The present study shows that cytosolic aldose reductase activity was maximal in the proximal and middle segments of the epididymis and decreased in the distal epididymis. Western and Northern blot analysis confirmed the distribution pattern of aldose reductase and of the encoding mRNA. The optimal pH of epididymal aldose reductase was 6.0-6.5 when glucose was used as a substrate; this corresponds to the pH of the intraluminal epididymal fluid. In order to evaluate the possible involvement of sorbitol in sperm physiology, Western blot of tissue homogenates were probed with an anti-sorbitol dehydrogenase antibody. The amount of enzyme immunodetected was higher in the proximal and distal segments of the epididymis when compared to the amount detectable in the middle segment of the epididymis. Sorbitol dehydrogenase activity as well as the level of the encoding mRNA showed the same pattern of distribution. Furthermore, immunohistological studies using the anti-sorbitol dehydrogenase revealed that this enzyme was synthesized by the epididymal epithelial cells bordering the intraluminal compartment. Knowing the importance of sorbitol and fructose in sperm metabolism, we hypothesized that the polyol pathway is involved in the modulation of sperm motility within the epididymis.

Aldose reductase and macrophage migration inhibitory factor are associated with epididymosomes and spermatozoa in the bovine epididymis.

During the epididymal transit, mammalian spermatozoa acquire new surface proteins necessary for male gamete function. We have previously shown that membranous vesicles, called epididymosomes, interact with spermatozoa allowing the transfer of some proteins to sperm surface within the epididymal lumen. The protein composition of those vesicles has been investigated to document the mechanisms of protein transfer from epididymosomes to spermatozoa. Electrophoretic analysis revealed that protein composition is different from the epididymal soluble compartment as well as from similar vesicles present in the semen. Protein association with epididymosome is very strong as revealed by resistance to extraction with detergent. Matrix-assisted laser desorption ionization time-of-flight as well as immunodetection techniques have been used to identify some proteins associated to epididymosomes and spermatozoa. An aldose reductase known for its 20alpha-hydroxysteroid dehydrogenase activity and the cytokine (macrophage migration inhibitory factor) have been identified. These two proteins have been immunolocalized in principal cells of the epididymal epithelium, a more intense signal being detected in the distal epididymal segment as well as in the vas deferens. Database search revealed that these two proteins are characterized by the lack of a signal peptide. These results are discussed with regard to a possible apocrine mode of secretion of these proteins acquired by spermatozoa during the epididymal transit.

Semen characteristics of genetically identical quadruplet bulls.

Modern cloning methods have become an important technology in artificial insemination which is used to create and maintain pools of genetically superior bull semen. In this study, semen from four identical quadruplet bulls (Q(1), Q(2), Q(3), and Q(4)) produced by blastomere separation was analyzed to evaluate the differences in reproductive potential, if any, that existed between the identical quadruplet siblings. Analysis of fresh semen collected from 1994 to 1996, showed lower progressive motility and lower sperm concentration for one bull (Q(3)) compared to his identical brothers (P<0.05). Semen characteristics following freezing-thawing procedures have also been tested for these quadruplet bulls. The percentage of motility, progressive motility, and mean path velocity were lower in Q(4) compared with Q(1). Moreover, intracellular calcium level and P25b level (P25b is a sperm surface protein proposed to be a potential bull fertility marker) were lower in Q(4) compared with his siblings (P<0.05). Cryodamage to Q(4)'s frozen-thawed spermatozoa were confirmed by a lower percentage of embryo development after in vitro fertilization. Thus, the higher instability of cryopreserved spermatozoa from Q(4) and the lower semen production of Q(3), compared to their siblings, indicate that differences in semen characteristics can indeed exist among genetically identical animals produced by blastomere separation.

Selected proteins of "prostasome-like particles" from epididymal cauda fluid are transferred to epididymal caput spermatozoa in bull.

During epididymal transit, spermatozoa acquire selected proteins secreted by epithelial cells. We recently showed that P25b, a protein with predictive properties for bull fertility, is transferred from prostasome-like particles present in the cauda epididymal fluid (PLPCd) to the sperm surface. To further characterize the interactions between PLPCd and epididymal spermatozoa, PLPCd were prepared by ultracentrifugation of bull epididymal fluid, then surface-exposed proteins were biotinylated and coincubated in different conditions with caput epididymal spermatozoa. Western blot analysis revealed that only selected proteins are transferred from PLPCd to spermatozoa. MALDI-TOF analysis revealed that these transferred proteins are closely related. The pattern of distribution of the PLPCd transferred varied from one sperm cell to the other, with a bias toward the acrosomal cap. This transfer appeared to be temperature sensitive, being more efficient at 32-37 degrees C than at 22 degrees C. Transfer of PLPCd proteins to spermatozoa was also pH dependant, the optimal pH for transfer being 6.0-6.5. The effect of divalent cations on PLPCd protein transfer to caput spermatozoa was investigated. Whereas Mg(2+) and Ca(2+) have no effect on the amount of proteins remaining associated with spermatozoa following coincubation, Zn(2+) had a beneficial effect. These results are discussed with regard to the function of PLPCd in epididymal sperm maturation.