In vitro fertility rate of 129 strain is improved by buserelin (gonadotropin-releasing hormone) administration prior to superovulation.

The 129 mice are well recognized for their low fertility and it is speculated that this lack of fertility may be due to the oocyte condition. In this study we investigated superovulation regimens for the 129S1/SvImJ mouse strain to improve the oocyte quality and fertility rate of in vitro fertilization (IVF). Female mice were divided into four groups based on hormone and timing of injection. Group 1 received pregnant mare serum gonadotropin (PMSG) and 48 h later human chorionic gonadotropin (hCG); using the same dose, group 2 received hCG 52 h post-PMSG and group 3, 55 h post-PMSG. Group 4 received buserelin (gonadotropin-releasing hormone agonist [GnRH]) followed 24 h later by PMSG and then hCG 55 h post-PMSG. IVF was performed using 129S1/SvImJ oocytes and sperm; C57BL/6J sperm with 129S1/SvImJ oocytes was used as fertility control. The IVF fertility rate was 1% (Groups 1 and 2), 17% (Group 3) and 55% (Group 4) for 129 oocytes fertilized with 129 sperm. For 129 oocytes fertilized with C57BL/6J sperm, the fertility rate was 5% (Group 1), 10% (Group 2), 40% (Group 3) and 59% (Group 4). These results suggest that extending the interval time between PMSG and hCG and giving GnRH in addition to the standard PMSG and hCG treatments can improve IVF fertility rate of 129S1/SvImJ mouse strains significantly.

Comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation.

Mouse sperm has proven to be more difficult to cryopreserve than sperm of other mammalian species. Published reports show that only three cryoprotectant agents (CPAs), alone or combined, have been studied: glycerol and dimethyl sulfoxide (DMSO), as permeating agents, and raffinose, as a nonpermeating agent. To date, the most consistent results for mouse sperm cryopreservation have been achieved by use of raffinose/skim milk as cryoprotectant with rapid cooling at 20 degrees C per minute. In this study, we compared the cryoprotection provided by permeating (glycerol, formamide, propanediol, DMSO, adonitol) or nonpermeating (lactose, raffinose, sucrose, trehalose, d-mannitol) compounds for freezing mouse sperm. Different solutions were made using 3% skim milk solution as the buffer or extender in which all different cryoprotectant agents were dissolved at a concentration of 0.3 M, with a final osmolality of approx. 400 mOsm. Sperm samples from CB6F1 (hybrid) and C57BL/6J (inbred) mice collected directly into each CPA were frozen/thawed under identical conditions. After thawing and CPA elimination (centrifugation) raffinose (59%), trehalose (61%), and sucrose (61%) sustained the best motility (P = < 0.1) of the nonpermeating agents, whereas the best of the permeating agents was DMSO (42%). Membrane integrity was analyzed and showed that the simple exposure (prefreeze) to sugars was less harmful than the exposure to glycols. Coincidentally, sperm frozen in trehalose (41%), raffinose (40.5%), and sucrose (37.5%) were the samples less injured among all different postthawed CPA tested. The in vitro fertilization results demonstrated that hybrid mouse spermatozoa frozen with sugars (lactose 80%, raffinose 80%, trehalose 79% of two-cell embryos production) were more fertile than those frozen with glycols (glycerol 11%).

In vitro fertilization with cryopreserved inbred mouse sperm.

Sperm from C57BL/6J, DBA/2J, BALB/cJ, 129S3/SvImJ, and FVB/NJ inbred mice were cryopreserved in 3% skim milk/18% raffinose cryoprotectant solution. The post-thaw sperm from all strains were evaluated for their viability and fertility by comparing them against B6D2F1 sperm used as a control. The protocol used for freezing mouse sperm was effective in different strains, because the motility was decreased by 50% after cryopreservation similar to other mammalian sperm. However, the progressive motility and the fertility of each inbred strain were affected differently. The C57BL/6J, BALB/cJ, and 129S3/SvImJ strains were the most affected; their fertility (two-cell cleavage) decreased from 70%, 34%, and 84% when using freshly collected sperm to 6%, 12%, and 6% when using frozen/thawed sperm, respectively. Live newborns derived from frozen/thawed sperm were obtained from all strains in the study. These results corroborate the genetic variation among strains with regard to fertility and susceptibility to cryopreservation.

Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: competence to undergo maturation, embryogenesis, and development to term.

Only primordial and primary follicles of frozen-thawed mouse ovaries survive after grafting to the ovarian bursa; large secondary follicles and antral follicles together with the oocytes contained in them degenerate. This study was undertaken to determine whether fully grown oocytes isolated from the antral follicles of frozen-thawed mouse ovaries are viable and can be rescued to undergo maturation, fertilization, and embryo development in vitro. Ovaries were cryopreserved after removal from 22-day-old (C57BL/6J x SJL/J)F(1) mice, with or without prior priming with equine chorionic gonadotrophin, and fresh non-frozen ovaries were used as controls. Only cumulus cell-denuded oocytes were recovered from frozen unprimed ovaries while both cumulus cell-enclosed and denuded oocytes were retrieved from frozen primed ovaries. Oocytes from both groups of frozen-thawed ovaries were able to undergo maturation, fertilization, and development to the blastocyst stage in vitro, though at lower percentages than oocytes from control unfrozen ovaries. Moreover, 19% of 2-cell stage embryos derived from frozen-thawed primed ovaries, compared with 42% of embryos derived from control primed ovaries, developed to term after transfer to pseudopregnant foster mothers (not significantly different). Therefore, fully grown oocytes in antral follicles survive the cryopreservation protocol, as demonstrated by maturation, fertilization and embryo development in vitro, and development to term after embryo transfer.

Expression of interferon-gamma in the lens exacerbates anterior uveitis and induces retinal degenerative changes in transgenic Lewis rats.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that has been implicated in immunopathogenic mechanisms of a number of inflammatory diseases of autoimmune or infectious disease etiology. However, its exact role is still a matter of debate. In experimental mouse models, IFN-gamma has been shown to exacerbate autoimmune thyroiditis, insulin-dependent diabetes mellitus, and autoimmune neuritis while it confers protection against experimental allergic encephalomyelitis and experimental uveitis. In this study, we generated transgenic rats with constitutive expression of IFN-gamma in the eye to study its paracrine effects and to investigate whether local production of IFN-gamma also confers protection against uveitis in the rat species. We show here that chronic exposure of ocular cells to IFN-gamma results in apoptotic death of retinal ganglion cells, development of chronic choroiditis, formation of retinal in-foldings, and activation of proinflammatory genes. In contrast to its protective systemic effect in the mouse, constitutive secretion of IFN-gamma in the rat eye was found to predispose the development of severe anterior uveitis and induction of retinal degenerative processes that impair visual acuity. Our data underscore the danger in extrapolation of cytokine effects in the mouse to humans without corroborating evidence in other species.

IFN-gamma increases the severity and accelerates the onset of experimental autoimmune uveitis in transgenic rats.

Experimental autoimmune uveitis (EAU) is a predominantly Th1-mediated intraocular inflammatory disease that serves as a model for studying the immunopathogenic mechanisms of uveitis and organ-specific autoimmune diseases. Despite the well-documented role of IFN-gamma in the activation of inflammatory cells that mediate autoimmune pathology, recent studies in IFN-gamma-deficient mice paradoxically show that IFN-gamma confers protection from EAU. Because of the implications of these findings for therapeutic use of IFN-gamma, we sought to reexamine these results in the rat, another species that shares essential immunopathologic features with human uveitis and is the commonly used animal model of uveitis. We generated transgenic rats (TR) with targeted expression of IFN-gamma in the eye and examined whether constitutive ocular expression of IFN-gamma would influence the course of EAU. We show here that the onset of rat EAU is markedly accelerated and is severely exacerbated by IFN-gamma. In both wild-type and TR rats, we found that the disease onset is preceded by induction of ICAM-1 gene expression and is characterized by selective recruitment of T cells expressing a restricted TCR repertoire in the retina. In addition, these events occur 2 days earlier in TR rats. Thus, in contrast to the protective effects of IFN-gamma in mouse EAU, our data clearly show that intraocular secretion of IFN-gamma does not confer protection against EAU in the rat and suggest that IFN-gamma may activate distinct immunomodulatory pathways in mice and rats during uveitis.

Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking.

Mouse half ovaries were cryopreserved and orthotopically transplanted into ovariectomized recipients genetically identical to ovary donors except for the coat color gene. Fertility was reestablished in 57% of the female recipients, which became pregnant in an average of 40 days after transplantation of frozen-thawed half ovaries. These experiments demonstrate that ovary cryopreservation can be a very useful option for banking mouse germplasm, or managing subfertile animal colonies, when embryo or sperm freezing cannot be used or is not cost effective.

Motility of cryopreserved mouse spermatozoa affected by temperature of collection and rate of thawing.

In an attempt to optimize a method of cryopreserving spermazoa from mice bearing mutations, we investigated the effect on motility of temperature for the collection of mouse sperm and the rate of thawing after freezing. Comparison among samples of sperm collected from both the caudae epididymides and vas deferens placed directly in a cryoprotectant of 3% skim milk and 18% raffinose equilibrated at 37, 23, and 3 degrees C showed no difference in the number of viable sperm harvested. Concentration and motility was highest after collection at 37 degrees C (22.3 x 10(6) sperm/ml with 80% motility) combined with rapid thawing at 37 degrees C (2.9 x 10(6) sperm/ml with 84% motility in the swim-up fraction). The fertilization capacity of sperm collected and thawed at 37 degrees C was analyzed in vitro and no difference was observed between the cryopreserved sperm and the control (91 and 89%, respectively). Transfer of in vitro fertilized embryos to pseudopregnant recipients resulted in 37% implantation at Day 10 of pregnancy and 38% live births at term.

Developmental changes and differential regulation by testosterone and estradiol of growth hormone receptor expression in the rabbit.

To investigate the effects of testosterone and estradiol (E2) on growth hormone receptor (GH-R) gene expression, we measured GH-R mRNA levels in relation to the changes of sex steroid concentrations in the normal male rabbits aged 1-12 months and after administration of testosterone or E2 to castrated male rabbits. In the normal animals, E2 levels were below the detection limit in all age groups, and testosterone levels were below the detection limit at 1 month, increased at 2 months and reached the plateau of the adult levels after 4 months. Liver GH-R mRNA levels were low at 1 month, reached a peak at 2 months and then decreased slightly thereafter. In the castrated animals, liver and growth plate GH-R mRNA levels were increased in the testosterone-treated group (162.0 +/- 12.0%, p < 0.025; 128.4 +/- 7.6%; p < 0.025) and reduced in the E2-treated group (29.6 +/- 6.2%, p < 0.005; 53.6 +/- 11.3%, p < 0.025). Sex steroid administration did not result in any significant change in GH-R mRNA levels in striated muscle, kidney and heart. Serum GH concentrations were increased in E2 (15.3 +/- 7.7 microg/l vs 4.8 +/- 2.2 microg/l, p < 0.025) but the increase was not significant in testosterone-treated animals (8.4 +/- 7.7 microg/l vs 4.8 +/- 2.2 microg/l). Both testosterone and E2 treatment resulted in a reduction of mean serum growth hormone-binding protein (GHBP) levels compared to control animals (1077 +/- 422 pmol/l, p < 0.01; 1137 +/- 443 pmol/l, p < 0.01; 2308 +/- 565 pmol/l). We conclude that in addition to their stimulatory effect on GH secretion, testosterone and E2 have opposite effects on GH-R gene expression in liver and growth plate in the rabbit. The modulation of GH-R expression by sex steroids may be important for growth during sexual maturation in mammals.

gamma Interferon expression disrupts lens and retinal differentiation in transgenic mice.

We previously generated an animal model for the study of autoimmune diseases of the eye by targeting gamma interferon (gamma IFN) expression to the lens of transgenic mice. Here, we have studied the effect of constitutive lens expression of gamma IFN on eye development of these transgenic mice. By Day 18 of embryonic development, lens and retinal differentiation programs are completely disrupted; normal lens epithelia and fibers are replaced by balloon-like cells and retinal differentiation into inner and outer neuroblastic layers is already affected. The mRNA levels of gamma E- and/or gamma F-crystallin and MIP, markers of lens cell differentiation, are drastically reduced, while expression of ICSBP, a gamma IFN-inducible transcriptional factor, is induced in the alpha ACry-gamma IFN transgenic mouse eyes. Taken together, our results suggest that constitutive expression of gamma IFN and its induction and activation of gamma IFN-inducible transcriptional factors in the eye altered the developmental fate of cells destined to become lens fiber cells by altering the pattern of lens gene expression.

Estradiol inhibits growth hormone receptor gene expression in rabbit liver.

We studied the ontogeny of GH receptor mRNA levels and the effect of exogenous estradiol administration on GH receptor mRNA levels in rabbit liver. A solution hybridization-RNase protection assay revealed a predominant 370-base long protected band corresponding to the mRNA encoding the transmembrane GH receptor, and a 241-base long protected band, representing about 9.0%, with the predicted size for the truncated form of the GH receptor. To study the developmental profile of GH receptor expression, we studied 12 female rabbits, at ages 1, 3, 5 and 7 months. Maximal GH receptor mRNA levels were observed in 3-month-old animals and decreased in 7-month-old animals. To investigate the effect of estradiol, 8-week-old immature female rabbits were randomly divided into five groups, and received subcutaneous pellets containing either placebo or estradiol at doses of 0.1, 0.5, 1.5 and 5.0 mg for 3 weeks. Exogenous administration of estradiol, at doses that resulted in physiological circulating levels, induced a reduction in GH receptor expression, measured both by GH binding (36 and 46%), and GH receptor mRNA levels (38 and 87%), in animals receiving pellets containing 1.5 and 5.0 mg of estradiol, respectively. We conclude that estradiol decreases GH receptor expression in rabbit liver. The results of our study suggest that there is an inverse relationship between circulating estrogen concentrations and liver GH receptor expression.

Ectopic expression of gamma interferon in the eyes of transgenic mice induces ocular pathology and MHC class II gene expression.

PURPOSE: To direct the expression of gamma IFN to the eyes of transgenic mice as a means of investigating the possible role of this lymphokine in ocular pathogenesis. METHODS: Transgenic mouse strains were generated by injection of a DNA fragment containing the murine alpha A-crystallin promoter fused to the coding sequence of murine gamma IFN gene. PCR and RT-PCR were used to screen for the presence of the transgene and mRNA analyses, respectively. Methacrylate-embedded eye sections were analyzed for morphology and cryosections for immunoperoxidase antibody staining. RESULTS: The most notable effects of gamma IFN in these transgenic mice include cataract, microphthalmia, blepharophimosis, microphakia, impairment of lens fiber formation, arrest of retinal differentiation, serous retinal detachment with presence of macrophages in the subretinal space, persistent hyperplastic primary vitreous, and corneal vascularization. MHC class II mRNA levels were significantly increased in the transgenic eyes and MHC class II proteins were expressed in their cornea, iris, ciliary body, choroid, lens and RPE. CONCLUSIONS: Ectopic expression of gamma IFN in the lens affected the growth of the whole eye, resulting in microphthalmia and microphakia. The author's data suggest that alpha ACry-gamma IFN transgenic mouse ocular cells express functional gamma IFN receptors and that interaction of gamma IFN with its receptor induced biochemical and morphologic changes in the transgenic eyes. These mice provide an animal model for the study of the linkage between aberrant MHC expression and predisposition to autoimmune diseases.