Immunocontraception is induced in BALB/c mice inoculated with murine cytomegalovirus expressing mouse zona pellucida 3

Immunocontraception, the prevention of oocyte fertilization through immunological means, could potentially be used to control plaguing mouse populations in Australia. This paper describes the construction of a mouse-specific betaherpesvirus, murine cytomegalovirus, which has been engineered to express the murine zona pellucida 3 (ZP3) gene. A single inoculation of this recombinant virus resulted in almost complete infertility, persistent anti-ZP3 antibody production, and profound changes to ovarian morphology in BALB/c mice in the absence of significant virus replication during the acute phase of infection. Murine cytomegalovirus may prove to be useful as a vector for the delivery of a mouse-specific immunocontraceptive agent to target populations of wild mice in the field.     

Maternal autoantibody triggers de novo T cell-mediated neonatal autoimmune disease

Although human maternal autoantibodies may transfer transient manifestation of autoimmune disease to their progeny, some neonatal autoimmune diseases can progress, leading to the loss of tissue structure and function. In this study we document that murine maternal autoantibody transmitted to progeny can trigger de novo neonatal pathogenic autoreactive T cell response and T cell-mediated organ-specific autoimmune disease. Autoantibody to a zona pellucida 3 (ZP3) epitope was found to induce autoimmune ovarian disease (AOD) and premature ovarian failure in neonatal, but not adult, mice. Neonatal AOD did not occur in T cell-deficient pups, and the ovarian pathology was transferable by CD4(+) T cells from diseased donors. Interestingly, neonatal AOD occurred only in pups exposed to ZP3 autoantibody from neonatal days 1-5, but not from day 7 or day 9. The disease susceptibility neonatal time window was not related to a propensity of neonatal ovaries to autoimmune inflammation, and it was not affected by infusion of functional adult CD4(+)CD25(+) T cells. However, resistance to neonatal AOD in 9-day-old mice was abrogated by CD4(+)CD25(+) T cell depletion. Finally, neonatal AOD was blocked by Ab to IgG-FcR, and interestingly, the disease was not elicited by autoantibody to a second, independent native ZP3 B cell epitope. Therefore, a new mechanism of neonatal autoimmunity is presented in which epitope-specific autoantibody stimulates de novo autoimmune pathogenic CD4(+) T cell response.     

Interferon-gamma contributes to the normalcy of murine pregnancy.

Uterine natural killer (uNK) cells are transient, large, heavily granulated, maternal lymphocytes present on the mesometrial side of the pregnant mouse uterus. These cells contribute to normal implantation site development. Cytokine production, particularly interferon (IFN)-gamma, is a major function of most NK cell subsets. In this study, uNK cells were assessed for IFN-gamma production. Local concentrations of IFN-gamma were measured in the mesometrial regions of murine implantation sites between Days 6 and 16 of gestation. IFN-gamma was detected by ELISA at all days studied in a random-bred (CD1) and an inbred (BALB/c) strain of immune-competent mouse and in two immune-deficient strains, SCID (NK(+), T(-), B(-)) and tgepsilon26 (NK(-), T(-), B(+)). Concentrations of IFN-gamma per implantation site peaked at Day 10 of gestation in NK(+) strains but were low and relatively constant in NK(-) mice. To evaluate the functions of IFN-gamma at murine implantation sites, pregnancy was studied in homozygously mated IFN-gamma(-/-) and IFN-gammaRalpha(-/-) mice and their congenic controls. Primiparous but not multiparous IFN-gamma(-/-) mice experienced significant fetal loss. Primiparous IFN-gammaRalpha(-/-) carried full litters to term. Implantation site pathology was demonstrated in both strains of gene-deleted mice by light microscopy and ultrastructurally. This included elevated numbers of uNK cells that contained fewer and smaller granules and, after Day 10 of gestation, progressive necrosis and loss of decidua. The presence of a fetus able to produce IFN-gamma did not modify the phenotype of pregnant IFN-gamma(-/-) mice. This study indicates that during murine pregnancy, uNK cells are the main source of IFN-gamma on the mesometrial side of the uterus and that IFN-gamma contributes to normal health of the midgestational decidua. Furthermore, evidence is presented that IFN-gamma-producing cells exist in mesometrial regions of implantation sites that are neither NK nor T cells.     

Development of dominant follicles and length of ovarian cycles in post-partum dairy cows

The resumption of ovarian activity after normal calvings was studied in 18 lactating Friesian cows. Since, in 17 cows, first post-partum ovulation occurred without overt oestrous behaviour being detected, the resultant cycles were called 'ovarian cycles'. The mean (+/- s.d.) length of the ovarian cycles was 21.0 +/- 8.7 days. The duration of cycles tended to be normal (18-24 days) or long (greater than or equal to 25 days) when the ovulatory dominant follicles were identified before Day 10 post partum; they were consistently short (9-13 days) when dominant follicles identified after Day 20 post partum ovulated. When such follicles were detected between Days 10 and 20 post partum, long, normal and short ovarian cycles were detected. The number of waves of follicular growth with associated dominant follicles observed during the ovarian cycles tended to be related to cycle length; short cycles had 1 dominant follicle, normal cycles predominantly 2, and long cycles mostly 3 dominant follicles. The mean (+/- s.d.) duration of 13 oestrous cycles studied was 23.1 +/- 2.1 days. Of these cycles, 7 had 3 and 6 had 2 dominant follicles. The oestrous cycles with 3 dominant follicles had a mean (+/- s.d.) duration of 24.0 +/- 1.2 days and the respective dominant non-ovulatory follicles reached maximum sizes on Days 8 and 18, respectively; oestrous cycles with 2 dominant follicles were 22.2 +/- 2.6 days in duration, and the dominant non-ovulatory follicle reached maximum size by Day 8. Ovarian follicular development during the first 45 days of pregnancy was characterized by the growth and regression of successive dominant follicles, each lasting 10-12 days. These results show that the first ovarian cycle was predominantly short when the ovulatory dominant follicle was first detected after Day 20 post partum.     

Retargeting T cell-mediated inflammation: a new perspective on autoantibody action

To understand the pathogenesis of organ-specific autoimmune disease requires an appreciation of how the T cell-mediated inflammation is targeted, and how the organ function is compromised. In this study, autoantibody was documented to influence both of these parameters by modulating the distribution of T cell-mediated inflammation. The murine autoimmune ovarian disease is induced by immunization with the ZP3330-342 peptide of the ovarian zona pellucida 3 glycoprotein, ZP3. Passively transferred or actively induced Ab to ZP3335-342 bound to the zona pellucida in the functional and degenerative ovarian follicles, and the ovaries remained histologically normal. Transfer of ZP3330-342 peptide-specific T cells targeted the degenerative follicles and spared the functional follicles, and the resultant interstitial oophoritis was associated with unimpaired ovarian function. Unexpectedly, the coexistence of ZP3330-342 peptide-specific T cells and zona-bound autoantibody led to a dramatic translocation of the ovarian inflammation to the growing and mature ovarian follicles, with destruction of the ovarian functional unit. Ab retargeted both Th1-induced mononuclear inflammation and Th2-induced eosinophilic inflammation, and retargeting was induced by murine and rat polyclonal Abs to multiple distinct native B cell determinants of the zona pellucida. Therefore, by reacting with the native determinants in tissue Ag, Ab alters the distribution of T cell-mediated inflammation, and results in destruction of the functional units of the target organ. We propose that this is a clinically important and previously unappreciated element of Ab action in autoimmune disease.     

Histological study of oogenesis pathology in smoky mutant mice

The various pathology of female mice reproductive system with the mutation smoky (smk) was observed. This recessive autosomal gene dilutes the black colour of hair to the light smoky. The cases with ovarian agenesis and dysgenesis of the mutants WR-smk/smk and B6-smk/smk without the germ cells or with the acute deficit was discovered. The oogenesis pathology of the mutants B6-smk/smk was revealed by the block of oocyte growth and their nucleus development in the growing nonatretic follicles leading to the female infertility. Endogene causes of the female hypogonadism and infertility associated with the mutation smoky are proposed.     

Persistent colonization and transient suppression of DTH responses in an estrogen-dependent vaginal candidosis murine model

Recurrence of vaginal candidosis in women of childbearing age has been attributed to several predisposing factors including the presence of significant amounts of estrogen in the reproductive tract. In this study, the effect of estrogen on the level of C. albicans colonization, persistence of infection and suppression of DTH responses was investigated in an estrogen-dependent vaginal candidosis murine model. Mice were first injected subcutaneously with 0.5 mg of estradiol valerate 72 hours prior to C. albicans intravaginal inoculation and at weekly intervals thereafter for a period of up to 4 weeks; the inoculum consisted of 2 x 10(7) stationary-phase C. albicans blastoconidia in a volume of 20 microl. C. albicans colonization was evaluated in the spleen, liver, kidney, small intestine and reproductive tract of estrogen-treated and control mice 72 hours following inoculation, DTH responses were evaluated 2 and 5 weeks following primary inoculation and persistence of infection was evaluated at days 2, 3, 4, 8, 12, 19 and 26 post inoculation. Estrogen-treated mice exhibited higher levels of C. albicans colonization compared with control mice; this was most evident in the small intestine and reproductive tract. Estrogen treatment resulted in pronounced suppression of C. albicans-specific DTH responses; in that average footpad swelling was 4.7 mm in untreated mice compared with 2.3 mm in estrogen-treated mice. Long-term estrogen treatment resulted in the persistence of infection; in contrast, C. albicans infection resolved by day 8 post inoculation in untreated mice. DTH responses assayed 5 weeks post primary inoculation in treated mice were on average 4.1 mm, this was similar to that observed in untreated mice tested for DTH response 2 and 5 weeks post primary inoculation. These results suggest that, on the one hand, estrogen has an enhancing effect on C. albicans colonization and persistence of infection. On the other, estrogen seems to suppress DTH responses within the first 2 weeks post infection; persistence of infection under the influence of estrogen, however, seems to coexist with detectable systemic cell-mediated immunity.     

Artesunate-induced depletion of embryonic erythroblasts precedes embryolethality and teratogenicity in vivo

BACKGROUND: Artesunate (ART), an artemisinin antimalarial, is embryolethal and teratogenic in rats, with the most sensitive days being 10 and 11 postcoitum (pc), respectively (Clark et al.: Birth Defects Res B 71:380-394, 2004; White et al.: Birth Defects Res A 70:265, 2004). METHODS: In this study, pregnant rats were administered a single oral dose of 17 mg/kg ART on Days 10-11 pc and conceptuses were evaluated through Day 14 pc. RESULTS: Paling of visceral yolk sacs was observed within 3-6 hr after treatment. Within 24 hr, marked paling and embryonic erythroblast depletion were observed macroscopically, which preceded malformations and embryo death, and persisted through Day 14 pc. Histologically, embryonic erythroblasts were reduced and cells showed signs of necrosis within 24 hr, were maximally depleted by 48 hr, and had partially rebounded within 3-4 days after treatment (Days 13 and 14 pc). Iron accumulation was evident in treated erythroblasts as early as 6 hr after treatment, suggesting impairment of heme synthesis. Heart abnormalities (swollen or collapsed chambers) were observed within 24 hr in approximately 25-60% of embryos and within 48 hr in 100% of embryos, correlating with histologic signs of cardiac myopathy (thinned and underdeveloped heart walls and enlarged chambers). Delays in limb and tail development occurred by Day 13 pc. Embryos were viable through Day 13 pc, but approximately 77% of embryos had died by Day 14 pc, presumably due to hypoxia and/or cardiac abnormalities. CONCLUSIONS: In summary, embryonic erythroblasts are the primary target of ART toxicity in the rat embryo after in vivo treatment, preceding embryolethality and malformations.     

Genetic susceptibility to chlamydial salpingitis and subsequent infertility in mice.

Groups of mice from genetically defined inbred strains were infected genitally with a pathogenic human strain of Chlamydia trachomatis and their subsequent fertility was compared. The CBA, C3H (H-2o) and C3H/He-mg (H-2k) mice were less fertile than control mice, at least up to 6 months after infection. In contrast, fertility was not impaired in BALB/c mice or in congenic BALB/K mice, which had the H-2k haplotype. Reduced fertility was paralleled by the extent of histological oviductal inflammation in mice of each strain. No salpingitis was seen 21 days after infection in the BALB strains, but lesions were apparent in CBA and C3H strains up to about 70 days after inoculation and these sometimes developed into hydrosalpinges. These results indicate that susceptibility to chlamydial salpingitis and subsequent infertility is under genetic control. This control was not simply associated with the major H-2 gene complex, as mouse strains of the same haplotype (H-2k) differed in susceptibility. The fertility of BALB/c (H-2d) and BALB/K (H-2k) strains was no different from that of controls, and congenic C3H mice of differing H-2 haplotypes (H-2k and H-2o) showed reduced fertility. Although all the infected F1 (BALB/K x C3H/He-mg) mice produced litters at the same rate as untreated controls, the litters were considerably smaller. This was due to the occurrence of unilateral pregnancies in the mice inoculated under the ovarian bursae and possibly also to early fetal death in mice inoculated directly in the uterus. These findings emphasize the importance of early diagnosis and treatment of infection of the lower genital tract of women.     

The follicle-deplete mouse ovary produces androgen

The follicle-depleted postmenopausal ovary is enriched in interstitial cells that produce androgens. This study was designed to cause follicle depletion in mice using the industrial chemical, 4-vinylcyclohexene diepoxide (VCD), and characterize the steroidogenic capacity of cells in the residual ovarian tissue. From a dose-finding study, the optimal daily concentration of VCD was determined to be 160 mg/kg. Female B6C3F(1) immature mice were treated daily with vehicle control or VCD (160 mg kg(-1) day(-1), 15 days, i.p.). Ovaries were removed and processed for histological evaluation. On Day 15 following onset of treatment, primordial follicles were depleted and primary follicles were reduced to about 10% of controls. On Day 46, primary follicles were depleted and secondary and antral follicles were reduced to 0.7% and 2.6% of control, respectively. Seventy-five percent of treated mice displayed disruptions in estrous cyclicity. All treated mice were in persistent diestrus (acyclic) by Day 58. Plasma FSH levels were increased (P < 0.05) relative to controls on Day 37 and had plateaued by Day 100. Relative to age-matched cyclic controls, by Day 127, the significant differences in VCD-treated mice included reduced ovarian and uterine weights, elevated plasma LH and FSH, and reduced plasma progesterone and androstenedione. Furthermore, plasma 17beta-estradiol levels were nondetectable. Unlike controls, immunostaining for LH receptor, and the high density lipoprotein receptor (SR-BI), was diffuse in ovarian sections from VCD-treated animals. Ovaries from Day 120 control and VCD-treated animals were dissociated and dispersed cells were placed in culture. Cultured cells from ovaries of VCD-treated animals produced less LH-stimulated progesterone than control cells. Androstenedione production was nondetectable in cells from cyclic control animals. Conversely, cells from VCD-treated animals produced androstenedione that was doubled in the presence of insulin and LH (1 and 3 ng/ml). Collectively, these data demonstrate that VCD-mediated follicle depletion results in residual ovarian tissue that may be analogous to the follicle-deplete postmenopausal ovary. This may serve as a useful animal model to examine the dynamics of follicle loss in women as ovarian senescence ensues.     

Persistence of physiological self antigen is required for the regulation of self tolerance

Endogenous Ag requirement for induction and maintenance of T cell tolerance has been extensively investigated in mice that express a transgenic Ag and/or its cognate transgenic TCR. In contrast, studies on tolerance for physiologically expressed self Ag and normal T cells are limited. Herein, we showed that the murine ovarian-specific ZP3 Ag is detectable from birth. Tolerance to ZP3 is detected in female relative to male mice. In comparison to males, 100-fold more ovarian peptide (pZP3) is required to elicit a comparable pathogenic response in females. Female tolerance to pZP3 was dependent on the presence of endogenous ovarian Ag, because neonatal ovariectomy converted the female response to that of males. Moreover, in female mice that were ovariectomized from the ages of 1-6 wk, the pZP3 responses were enhanced to the male level if ovaries were removed up to 7 days, but not 3 days, before adult challenge with pZP3. Thus, the physiologically expressed ZP3 Ag induces tolerance to pZP3, and the maintenance of tolerance is critically dependent on the continuous presence of the endogenous ovarian Ag. In contrast, exposure to endogenous ovarian Ag confined to the neonatal period is insufficient for the induction and maintenance of tolerance to ZP3.     

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.     

Autoimmune ovarian inflammation triggered by proinflammatory (Th1) T cells is compatible with normal ovarian function in mice.

The detection of noninfectious ovarian inflammation (oophoritis) and serum ovarian autoantibodies in a patient with premature ovarian failure is indicative of an autoimmune etiology. The mechanisms of autoimmune ovarian injury leading to loss of function are currently unknown. In this study we investigated the impact of oophoritis on ovarian function based on two murine autoimmune ovarian disease (AOD) models. AOD can be induced by thymectomy at Day 3 after birth (d3tx). D3tx mice develop ovarian inflammation and atrophy with loss of oocytes. In these mice, ovarian atrophy and not oophoritis correlated with abnormal estrous cyclicity. The second AOD model is induced by active immunization of adult mice with a murine ZP3 peptide (pZP3) in adjuvant. After active immunization, the zona pellucida antibody titer, not oophoritis, correlated with reduced fertility. To investigate the effect of oophoritis in the absence of antibody response or ovarian atrophy, pZP3-specific T cells were passively transferred into naive syngeneic mice. This recruited cytokine-producing cells into the ovaries so that elevated cytokine production and its effect on ovarian function could be examined. Recipients of pZP3-specific T cells developed severe granulomatous oophoritis, and the diseased ovaries had elevated ovarian mRNA levels of interferon-gamma, interleukin-1beta, and tumor necrosis factor alpha. Despite these changes, fertility rates and gonadotropin-induced follicular development remained essentially normal. Therefore, normal ovarian function is compatible with severe ovarian inflammation mediated by autoreactive T cells.     

Relationships between ovarian blood flow and ovarian response to eCG-treatment of dairy cows

The goal of the present study was to investigate ovarian blood flow and ovarian response in cows undergoing a gonadotropin treatment to induce a superovulatory response, using transrectal colour Doppler sonography. Forty-two cows including 19 cross-bred, 14 German Holstein and 9 German Black Pied cows were examined sonographically before hormonal stimulation on Day 10 of the oestrous cycle, three days after administration of eCG (Day 13) and seven days after artificial insemination (Day 7(p.i.)). After each Doppler examination, blood was collected for determination of total oestrogens (E) and progesterone (P4) in peripheral plasma. The blood flow volume (BFV) and pulsatility index (PI), which is a measure for blood flow resistance, were determined in the ovarian artery, and B-mode sonography was used to count dominant follicles and corpora lutea. Important criteria to assess the ovarian response following the hormonal treatment were the number of follicles >5mm in diameter on Day 13 and the number of corpora lutea on Day 7(p.i.) per cow. The number of follicles ranged from 2 to 61 (mean+/-S.E.M.: 17.5+/-1.7) and corpora lutea from 0 to 50 (mean+/-S.E.M.: 17.0+/-1.6). The BFV increased from 28.4 to 45.0 ml/min between Days 10 and 13 and reached a maximum of 108.5 ml/min on Day 7(p.i.) The PI decreased from 6.25 on Day 10 to 4.70 on Day 13 and to 2.10 on Day 7(p.i.) The BFV and PI on Day 13 did not correlate with the number of follicles (P>0.05). However, on Day 7(p.i.) the number of corpora lutea correlated positively with the BFV (r=0.64; P<0.0001), and an inverse relationship was found for the PI (r=-0.51; P=0.0005). There were no correlations (P>0.05) between the BFV and PI on Day 10 and the number of follicles on Day 13 or the number of corpora lutea on Day 7(p.i.) Results of the present study show that in cows, a hormonal treatment to induce a superovulatory response yielded a marked increase in BFV and a marked decrease in PI in the ovarian artery. However, there was no correlation between BFV and PI in the ovarian arteries before hormonal stimulation and the number of follicles and corpora lutea that developed after stimulation. Thus BFV and PI measured in the ovarian arteries have limited diagnostic value to predict the outcome of a gonadotropin treatment.     

Ovarian follicular populations during early pregnancy in heifers

Ovarian follicles >/=2mm were studied in 14 pregnant and 14 nonpregnant Holstein heifers by daily ultrasound examinations. There were significant differences among days, from Day 0 (day of ovulation) to Day 21, in the diameter of the largest follicle and the diameter of the second largest follicle in pregnant and nonpregnant heifers. There was an interaction of day and reproductive status (P < 0.001) for the diameter of the largest follicle. Significant differences among days were also observed in the numbers of follicles 2 to 3 mm, 4 to 6 mm, 7 to 10 mm, 11 to 13 mm, and >13 mm, and the total number of follicles >/=2 mm. There was a significant main effect of reproductive status for the number of follicles 11 to 13 mm. An interaction of day and reproductive status was observed for the number of follicles >13 mm, but not for any of the other diameter categories. The effect of reproductive status for number of follicles 11 to 13 mm and the interactions for the number of follicles >13 mm and the diameter of the largest follicle seemed due to the selective growth and ovulation of the follicle destined to ovulate in nonpregnant heifers. The differences in ovarian follicular populations between pregnant and nonpregnant heifers were attributed solely to the presence of a physiological mechanism for the selection of an ovulatory follicle in nonpregnant heifers. There were no significant differences among days for any follicular endpoint during Days 22 to 60 in the pregnant heifers.     

Cocaine enhances myocarditis induced by encephalomyocarditis virus in murine model

This study was designed to investigate whether cocaine can exacerbate viral myocarditis and increase its incidence. Recent clinical evidence suggests that cocaine abuse increases the incidence of myocarditis. However, it has not been directly demonstrated that cocaine exposure enhances murine myocarditis. BALB/c mice were divided into eight groups: saline control, encephalomyocarditis virus (EMCV), 10 mg/kg cocaine (Coc-10), 30 mg/kg cocaine (Coc-30), 50 mg/kg cocaine (Coc-50), EMCV+Coc-10, EMCV+Coc-30, EMCV+Coc-50. After inoculation with EMCV, the mice were treated daily with either cocaine or saline for 90 days. Mice were euthanized at different days after EMCV inoculation. Mortality was recorded and myocarditis severity was evaluated. The mortality of the myocarditis mice treated with cocaine increased significantly, from 22% (EMCV) to 25.7% (Coc-10+EMCV), 41.4% (Coc-30+EMCV), and 51.4% (Coc-50+EMCV) (P < 0.05), respectively. The incidence and severity of inflammatory cell infiltration and myocardial lesions was higher in infected mice exposed to cocaine. Cocaine administered only before infection did not exacerbate myocarditis. Norepinephrine (NE) assay showed that cocaine exposure significantly increased myocardial NE concentration but this increase was partially inhibited in infected animals. Adrenalectomy abolished the effect of cocaine on mortality. Furthermore, propranolol, a beta-blocker, significantly decreased the enhancing effects of cocaine on myocarditis mice. In conclusion, cocaine increases the severity and mortality of viral myocarditis in mice. Increased catecholamines may be a major factor responsible for this effect.     

Analysis of the contraceptive potential of antibodies against native and deglycosylated porcine ZP3 in vivo and in vitro.

We have undertaken a comparative analysis of the contraceptive activity of antibodies directed against the porcine sperm receptor zona pellucida antigen (ZP3) and its Mr = 32,000 polypeptide core (DGZP-32). The strategies employed for this analysis included the induction of active immunity in a primate, the common marmoset, and an in vitro fertilization protocol involving the use of viable human ova. In both experimental situations, antibodies against ZP3 were shown to exhibit contraceptive activity, leading respectively to the induction of long-term infertility in the primate model and to the complete inhibition of human fertilization in vitro. The in vivo studies also revealed that the induction of high titer antibodies against ZP3 was inevitably associated with the appearance of an ovarian pathology characterized by the progressive depletion of the primordial follicle pool within one to two years. This side effect could not be alleviated by the use of DGZP-32 as antigen since the induction of immunity against this polypeptide was also associated with the eventual appearance of an ovarian pathology identical to that observed with ZP3. Furthermore, the DGZP-32 peptide was less effective than ZP3 in inducing the formation of antibodies capable of inhibiting the fertilization of human ova in vitro. We conclude that significant problems remain with the use of deglycosylated zona peptides for the development of contraceptive vaccines and that their potential will not be realized until the epitopes responsible for the induction of infertility and the primordial follicle depletion have been identified and segregated.     

Uterine involution and ovarian changes during early post partum in autumn-lambing Corriedale ewes

The time of uterine involution and the changes in ovarian follicle populations were studied during early postpartum in multiparous, suckling Corriedale ewes lambing in the autumn. On Day 1 (n=5), Day 5 (n=4), Day 17 (n=4) and Day 30 (n=3) postpartum ewes underwent surgery to obtain ovaries and uteri. The weights of uteri and the lengths of the previous pregnant and nonpregnant horns were recorded as well as the presence of ovarian follicles larger than 1 mm in diameter. Uterine weight and length of uterine horns decreased (P2 to < 4 mm) follicles were also present. At days 17 and 30, aside from the small and medium follicles, all the ewes also had large (>/= 4 mm) follicles, and, at Day 30 2 ewes had large corpora lutea. We conclude that in autumn-lambing Corriedale ewes macroscopic uterine involution was complete around Day 17 post partum and that follicle development begins immediately after parturition, reaching preovulatory size before Day 17. In 2 of the 3 ewes studied until Day 30, ovulation (first progesterone increase) occurred after Day 17 (Days 18 and 25).     

Adoptive transfer of dendritic cells modulates immunogenesis and tolerogenesis in a neonatal model of murine cutaneous leishmaniasis

We evaluated the adoptive transfer of DCs on Leishmania (L.) mexicana-infected neonatal BALB/c mice. DCs were isolated and purified from the spleens of the following donor groups: a) Adult BALB/c mice infected during adulthood with L. (L) mexicana; b) Adult BALB/c mice infected during neonatal life; c) Healthy neonatal BALB/c mice; d) Healthy adult BALB/c mice. A neonatal model of infection, generated after inoculation with 5 × 10⁵ promastigotes of L. (L) mexicana, was used as the infection control group. Sixteen hours after intraperitoneal transfer of DCs (1 × 10³, 1 × 10⁵, or 1 × 10⁶ cells/ml), neonatal recipient BALB/c mice were infected. The adoptive transfer of DCs diminished disease progression in neonatal mice. This reduction depends on the quantity and provenance of transferred DCs, since the effect was more evident with high numbers of DCs from adult mice infected during adulthood and healthy neonatal mice. Protection was significantly reduced in animals receiving DCs from healthy adult mice but it was absent in mice receiving DCs from adult mice infected during neonatal life. These results suggest that genetic susceptibility to Leishmania infection can be modified during neonatal life, and that the period of life when antigens are encountered is crucial in influencing the capacity of DCs to induce resistance or tolerance.