Inhibition of fertility in mice by passive immunization with antibodies to isolated zonae pellucidae.

A chromatographically purified preparation of gamma globulin produced against isolated zonae pellucidae of mice was used to immunize mice. A single 2.5 mg dose totally inhibited fertility for a minimum of 11 days.     

Human sperm bind to the N-terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice

Fertilization requires taxon-specific gamete recognition, and human sperm do not bind to zonae pellucidae (ZP1-3) surrounding mouse eggs. Using transgenesis to replace endogenous mouse proteins with human homologues, gain-of-function sperm-binding assays were established to evaluate human gamete recognition. Human sperm bound only to zonae pellucidae containing human ZP2, either alone or coexpressed with other human zona proteins. Binding to the humanized matrix was a dominant effect that resulted in human sperm penetration of the zona pellucida and accumulation in the perivitelline space, where they were unable to fuse with mouse eggs. Using recombinant peptides, the site of gamete recognition was located to a defined domain in the N terminus of ZP2. These results provide experimental evidence for the role of ZP2 in mediating sperm binding to the zona pellucida and support a model in which human sperm-egg recognition is dependent on an N-terminal domain of ZP2, which is degraded after fertilization to provide a definitive block to polyspermy.     

Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss.

All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (Zp1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing Zp1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some Zp1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from Zp1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from Zp1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.     

Large scale isolation of bovine and pig zonae pellucidae: Chemical,immunological, and receptor properties

A procedure is described for isolating milligram quantities of bovine and porcine zonae pellucidae, uncontaminated by follicle cells or their processes. On SDS-polyacrylamide gel electrophoresis the isolated bovine zona material formed one major glycoprotein band with an estimated molecular weight of approximately 100,000 daltons and two minor lower molecular weight components. The isolated pig zonae formed only one glycoprotein band with a molecular weight of approximately 62,000 daltons. Rabbit antisera raised against the isolated zonae were zona-specific and formed only a single precipitin line against the heat-solubilized zonae on immunoelectrophoresis. An adjuvant was not required for high-antibody titers. High titers were also obtained by injection of the dog and rhesus monkey. Anti-zona antibody was detected by immunofluorescence, zona-coating, double-immunodiffusion, and the inhibition of spermbinding to eggs, including those of human origin. Antigenic and sperm receptor properties were stable at 100°C for five minutes, but some activity was lost after longer exposure. The serum antibody produced by rabbits immunized with pig zonae was predominantly IgG and cross-reacted with the zonae of a variety of other species, including primates. Pregnancy was inhibited in female rabbits immunized with pig zona preparations.     

Properties of intact and univalent (Fab) antibodies raised against isolated, solubilized, mouse zonae pellucidae

Intact and univalent antibodies were prepared against mechanically isolated mouse zonae pellucidae solubilized in a variety of ways (heat, low pH, SDS, urea and trypsin). The antisera bound avidly and specifically to solubilized iodinated zona antigens and the intact zona structure. When the concentrations of immunoreactive Fab material in the intact and univalent antibody preparations were equalized and compared for their ability to block the sperm-binding stage of fertilization, only the intact gamma-globulin preparations possessed antifertility activity. These results indicate that antibodies raised against intact solubilized zonae pellucidae block fertilization by cross-linking antigens on the outer zona surface, thereby indirectly masking the sperm receptor sites. The integrity of these surface components did not appear to be affected by solubilization procedures that disrupt non-covalent bonds (heating, low pH, SDS and urea) although they did appear to be adversely affected by trypsin treatment. None of the antisera tested contained antibodies directed against the sperm receptor site indicating that these critical components lack immunogenicity.     

Defective smooth muscle development in qkI-deficient mice

The qkI gene encodes an RNA binding protein which was identified as a candidate for the classical neurologic mutation, qkv. Although qkI is involved in glial cell differentiation in mice, qkI homologues in other species play important roles in various developmental processes. Here, we show a novel function of qkI in smooth muscle cell differentiation during embryonic blood vessel formation. qkI null embryos died between embryonic day 9.5 and 10.5. Embryonic day 9.5 qkI null embryos showed a lack of large vitelline vessels in the yolk sacs, kinky neural tubes, pericardial effusion, open neural tubes and incomplete embryonic turning. Using X-gal and immunohistochemical staining, qkI is first shown to be expressed in endothelial cells and smooth muscle cells. Analyses of qkI null embryos in vivo and in vitro revealed that the vitelline artery was too thin to connect properly to the yolk sac, thereby preventing remodeling of the yolk sac vasculature, and that the vitelline vessel was deficient in smooth muscle cells. Addition of QKI and platelet-endothelial cell adhesion molecule-1 positive cells to an in vitro para-aortic splanchnopleural culture of qkI null embryos rescued the vascular remodeling deficit. These data suggest that QKI protein has a critical regulatory role in smooth muscle cell development, and that smooth muscle cells play an important role in inducing vascular remodeling.     

Effect of antibody to detergent solubilized zonae pellucidae on in vivo and in vitro fertilization in the mouse

An antiserum was produced in one rabbit against mouse zonae pellucidae solubilized with 70 mM Na₂SO₃, 1% SDS, and 0.04 mM CuSO₄. An IgG of zona antibody completely inhibited fertilization both in vitro and in vivo in the mouse. F(ab′)₂ fragment obtained by pepsin digestion of IgG zona antibody inhibited fertilizability of eggs in vitro but did not inhibit fertilization in vivo after passive immunization.     

Defective vascular development in connexin 45-deficient mice

In order to reveal the biological function(s) of the gap-junction protein connexin 45 (Cx45), we generated Cx45-deficient mice with targeted replacement of the Cx45-coding region with the lacZ reporter gene. Heterozygous Cx45(+/)(-) mice showed strong expression of the reporter gene in vascular and visceral smooth muscle cells. Cx45-deficient embryos exhibited striking abnormalities in vascular development and died between embryonic day (E) 9.5 and 10.5. Differentiation and positioning of endothelial cells appeared to be normal, but subsequent development of blood vessels revealed impaired formation of vascular trees in the yolk sac, impaired allantoic mesenchymal ingrowth and capillary formation in the labyrinthine part of the placenta, and arrest of arterial growth, including a failure to develop a smooth muscle layer surrounding the major arteries of the embryo proper. As a consequence, the hearts of most Cx45-deficient embryos were dilated. The abnormal development of the vasculature in the yolk sac of Cx45(-)(/)(-) embryos could be caused by defective TGFbeta signalling, as the amount of TGF beta1 protein in the epithelial layer of the yolk sac was largely decreased in the E9.5 Cx45(-)(/)(-) embryo, compared with the wild-type embryo. The defective vascular development was accompanied by massive apoptosis, which began in some embryos at E8.5 and was abundant in virtually all tissues of the embryos at E9.5. We conclude that in Cx45(-)(/)(-) embryos, vasculogenesis was normal, but subsequent transformation into mature vessels was interrupted. Development of different types of vessels was impaired to a varying extent, which possibly reflects the complementation by other connexin(s).     

Structural and viral association comparisons of bovine zonae pellucidae from follicular oocytes, Day-7 embryos and Day-7 degenerated ova

Bovine zonae pellucidae (ZP) from follicular oocytes and from embryos and degenerated ova collected on Day 7 from superovulated cows were examined by scanning electron microscopy, by dimensional measurement, and by total protein determination. The number of plaque-forming units (PFU) of infectious bovine rhinotracheitis virus (IBRV) that were associated with ZP-intact embryos/ova from each of the 3 sources after in vitro exposure was also determined.

Scanning electron microscopy revealed that the surfaces of Day-7 embryos and degenerated ova were smoother than those of follicular oocytes. Mean dimensional measurements of the diameter/thickness of the ZP from follicular oocytes, Day-7 embryos, and degenerated Day-7 ova were 156.7 μm/12.3μm, 161.3μm/12.6μm, and 158.9μm/12.8μm, respectively. The mean total protein per ZP of follicular oocytes, embryos, and degenerated ova was 0.331 μg, 0.349 μg, and 0.254 μg, respectively. Considerable variability existed within groups, but significantly greater quantities of IBRV were associated with follicular oocytes (mean PFU/oocyte = 68.1) than with Day-7 embryos (mean PFU/embryo = 43.0; P<0.05) or with Day-7 ova (mean PFU/ovum = 31.9; P<0.01).

The reliability of using an assay for IBRV associated with nontransferable ova/embryos as an indicator of the presence or absence of the virus in transferable embryos from the same collection (Day 7) was supported. Although structural differences between the ZPs of follicular oocytes and Day-7 embryos were observed in this study, further investigation is needed to determine if there are differences in the protective function of the respective ZPs.     

Selective cortical interneuron and GABA deficits in cyclin D2-null mice

In contrast to cyclin D1 nulls (cD1-/-), mice without cyclin D2 (cD2-/-) lack cerebellar stellate interneurons; the reason for this is unknown. In the present study in cortex, we found a disproportionate loss of parvalbumin (PV) interneurons in cD2-/- mice. This selective reduction in PV subtypes was associated with reduced frequency of GABA-mediated inhibitory postsynaptic currents in pyramidal neurons, as measured by voltage-clamp recordings, and increased cortical sharp activity in the EEGs of awake-behaving cD2-/- mice. Cell cycle regulation was examined in the medial ganglionic eminence (MGE), the major source of PV interneurons in mouse brain, and differences between cD2-/- and cD1-/- suggested that cD2 promotes subventricular zone (SVZ) divisions, exerting a stronger inhibitory influence on the p27 Cdk-inhibitor (Cdkn1b) to delay cell cycle exit of progenitors. We propose that cD2 promotes transit-amplifying divisions in the SVZ and that these ensure proper output of at least a subset of PV interneurons.     

Normal sexual development and fertility in testatin knockout mice

The testatin gene was previously isolated in a screen focused on finding novel signaling molecules involved in sex determination and differentiation. testatin is specifically upregulated in pre-Sertoli cells in early fetal development, immediately after the onset of Sry expression, and was therefore considered a strong candidate for involvement in early testis development. testatin expression is maintained in the adult Sertoli cell, and it can also be found in a small population of germ cells. Testatin shows homology to family 2 cystatins, a group of broadly expressed small secretory proteins that are inhibitors of cysteine proteases in vitro but whose in vivo functions are unclear. testatin belongs to a novel subfamily among the cystatins, comprising genes that all show expression patterns that are strikingly restricted to reproductive tissue. To investigate a possible role of testatin in testis development and male reproduction, we have generated a mouse with targeted disruption of the testatin gene. We found no abnormalities in the testatin knockout mice with regard to fetal and adult testis morphology, cellular ultrastructure, body and testis weight, number of offspring, spermatogenesis, or hormonal parameters (testosterone, luteinizing hormone, and follicle-stimulating hormone).     

Defective dendrite elongation but normal fertility in mice lacking the Rho-like GTPase activator Dbl

Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation.     

Kindlin-2 in Sertoli cells is essential for testis development and male fertility in mice

Kindlin-2 is known to play important roles in the development of mesoderm-derived tissues including myocardium, smooth muscle, cartilage and blood vessels. However, nothing is known for the role of Kindlin-2 in mesoderm-derived reproductive organs. Here, we report that loss of Kindlin-2 in Sertoli cells caused severe testis hypoplasia, abnormal germ cell development and complete infertility in male mice. Functionally, loss of Kindlin-2 inhibits proliferation, increases apoptosis, impairs phagocytosis in Sertoli cells and destroyed the integration of blood-testis barrier structure in testes. Mechanistically, Kindlin-2 interacts with LATS1 and YAP, the key components of Hippo pathway. Kindlin-2 impedes LATS1 interaction with YAP, and depletion of Kindlin-2 enhances LATS1 interaction with YAP, increases YAP phosphorylation and decreases its nuclear translocation. For clinical relevance, lower Kindlin-2 expression and decreased nucleus localization of YAP was found in SCOS patients. Collectively, we demonstrated that Kindlin-2 in Sertoli cells is essential for sperm development and male reproduction.Subject terms: Apoptosis, Male factor infertility     

Genetic modifiers of the Kv beta2-null phenotype in mice

Shaker-type potassium (K+) channels are composed of pore-forming alpha subunits associated with cytoplasmic beta subunits. Kv beta2 is the predominant Kv beta subunit in the mammalian nervous system, but its functions in vivo are not clear. Kv beta2-null mice have been previously characterized in our laboratory as having reduced lifespans, cold swim-induced tremors and occasional seizures, but no apparent defect in Kv alpha-subunit trafficking. To test whether strain differences might influence the severity of this phenotype, we analyzed Kv beta2-null mice in different strain backgrounds: 129/SvEv (129), C57BL/6J (B6) and two mixed B6/129 backgrounds. We found that strain differences significantly affected survival, body weight and thermoregulation in Kv beta2-null mice. B6 nulls had a more severe phenotype than 129 nulls in these measures; this dramatic difference did not reflect alterations in seizure thresholds but may relate to strain differences we observed in cerebellar Kv1.2 expression. To specifically test whether Kv beta1 is a genetic modifier of the Kv beta2-null phenotype, we generated Kv beta1.1-deficient mice by gene targeting and bred them to Kv beta2-null mice. Kv beta1.1/Kv beta2 double knockouts had significantly increased mortality compared with either single knockout but still maintained surface expression of Kv1.2, indicating that trafficking of this alpha subunit does not require either Kv beta subunit. Our results suggest that genetic differences between 129/SvEv and C57Bl/6J are key determinants of the severity of defects seen in Kv beta2-null mice and that Kv beta1.1 is a specific although not strain-dependent modifier.     

Inhibition of urethane-induced genotoxicity and cell proliferation in CYP2E1-null mice

Urethane is a multi-site animal carcinogen and was classified as "reasonably anticipated to be a human carcinogen." Urethane is a fermentation by-product and found at appreciable levels in alcoholic beverages and foods such as bread and cheese. Recent work in this laboratory demonstrated for the first time that CYP2E1 is the principal enzyme responsible for urethane metabolism. The current studies were undertaken to assess the relationships between CYP2E1-mediated metabolism and urethane-induced genotoxicity and cell proliferation as determined by induction of micronucleated erythrocytes (MN) and expression of Ki-67, respectively, using CYP2E1-null and wild-type mice. Urethane was administered at 0 (vehicle), 1, 10, or 100mg/kg/day (p.o.), 5 days/week for 6 weeks. A significant dose-dependent increase in MN was observed in wild-type mice; however, a slight increase was measured in the MN-polychromatic erythrocytes in CYP2E1-null mice treated with 100mg/kg. A significant increase in the expression of Ki-67 was detected in the livers and the lungs (terminal bronchioles, alveoli, and bronchi) of wild-type mice administered 100mg urethane/kg in comparison to controls. In contrast, CYP2E1-null mice administered this dose exhibited negligible alterations in Ki-67 expression in the livers and lungs compared to controls. Interestingly, while Ki-67 expression in the forestomach decreased in wild-type mice, it increased in CYP2E1-null mice. Subsequent comparative metabolism studies demonstrated that total urethane-derived radioactivity in the plasma, liver, and lung was significantly higher in CYP2E1-null versus wild-type mice and un-metabolized urethane constituted greater than 83% of the radioactivity in CYP2E1-null mice. Un-metabolized urethane was not detectable in the plasma, liver, and lung of wild-type mice. In conclusion, these data demonstrated that CYP2E1-mediated metabolism of urethane, presumably via epoxide formation, is necessary for the induction of genotoxicity, and cell proliferation in the liver and lung of wild-type mice.     

Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice

Oocyte-specific deletion of ovarian genes using Cre/loxP technology provides an excellent tool to understand their physiological roles during folliculogenesis, oogenesis, and preimplantation embryonic development. We have generated a transgenic mouse line expressing improved Cre recombinase (iCre) driven by the mouse growth differentiation factor-9 (GDF-9) promoter. The resulting transgenic mouse line was named GDF-9-iCre mice. Using the floxed ROSA reporter mice, we found that Cre recombinase was expressed in postnatal ovaries, but not in heart, liver, spleen, kidney, and brain. Within the ovary, the Cre recombinase was exclusively expressed in the oocytes of primordial follicles and follicles at later developmental stages. The expression of iCre of GDF-9-iCre mice was shown to be earlier than the Cre expression of Zp3Cre and Msx2Cre mice, in which the Cre gene is driven by zona pellucida protein 3 (Zp3) promoter and a homeobox gene Msx2 promoter, respectively, in the postnatal ovary. Breeding wild-type males with heterozygous floxed germ cell nuclear factor (GCNF) females carrying the GDF-9-iCre transgene did not produce any progeny having the floxed GCNF allele, indicating that complete deletion of the floxed GCNF allele can be achieved in the female germline by GDF-9-iCre mice. These results suggest that GDF-9-iCre mouse line provides an excellent genetic tool for understanding functions of oocyte-expressing genes involved in folliculogenesis, oogenesis, and early embryonic development. Comparison of the ontogeny of the Cre activities of GDF-9-iCre, Zp3Cre, and Msx2Cre transgenic mice shows there is sequential Cre activity of the three transgenes that will allow inactivation of a target gene at different points in folliculogenesis.