John R. Morrison

Developmental Expression of Thyroid Hormone Receptors in the Rat Testis

Abstract Sertoli cell proliferation in the rat is completed by Days 15–20 postnatally. Thyroid hormones appear to regulate the duration of Sertoli cell proliferation, affecting adult Sertoli cell number and hence the capacity of the testis to produce sperm. In the present study, a combination of immunohistochemistry, immunoblot analysis, and reverse transcription-polymerase chain reaction was used to demonstrate the expression pattern of thyroid hormone receptors (TR) in the juvenile and adult rat testis. The results indicated that TRα1 was expressed in proliferating Sertoli cell nuclei, its expression decreasing coincident with the cessation of proliferation. TRα2, TRα3, and TRβ1 mRNAs were expressed at low levels during development; however, the corresponding protein was not detected by immunoblot analysis. In addition, TRα1 was found to be expressed in germ cells from intermediate spermatogonia to mid-cycle pachytene spermatocytes. Immunohistochemistry also demonstrated TR expression in a subset of interstitial cells. The demonstration of TR expression in germ cells undergoing spermatogenic differentiation suggests a possible role for thyroid hormones in the adult testis.

The role of activin, follistatin and inhibin in testicular physiology.

The role of the inhibins, activins and follistatins in testicular function are being more clearly defined following studies describing the cellular localisation of these proteins to the testis and the availability of specific assay systems enabling measurement of these proteins. Taken together with the results of targetted gene inactivation experiments, several concepts emerge. Inhibin B is predominantly produced by the Sertoli cell in many adult male mammals whereas there is a perinatal peak of inhibin A in the rat. In contrast, activin A has its highest concentrations in the immediate post-natal period during which it is involved in the developmental regulation of both germ cells and Sertoli cells being modulated by follistatin. Activin A levels are considerably lower in the adult testis but Sertoli cell production is stimulated by interleukin-1 and inhibited by FSH. Little is known about the production of activin B due to the absence of a suitable assay but the beta(B) subunit mRNA is expressed in germ cells and Sertoli cells and is stage-dependent. This pattern of expression suggest that it may be involved in autocrine or paracrine actions within the seminiferous epithelium.

FGFR-1 [corrected] signaling is involved in spermiogenesis and sperm capacitation.

Cloning of the fibroblast growth factor receptor (FGFR) adaptor Snt-2 cDNA and the identification of FGFR-1 protein in association with sperm tails, suggested that FGFR-1 signaling was involved in either sperm tail development or function. This hypothesis was tested by the creation of transgenic mice that specifically expressed a dominant-negative variant of FGFR-1 in male haploid germ cells. Mating of transgenic mice showed a significant reduction in pups per litter compared with wild-type littermates. Further analysis demonstrated that this subfertility was driven by a combination of reduced daily sperm output and a severely compromised ability of those sperm that were produced to undergo capacitation prior to fertilization. An analysis of key signal transduction proteins indicated that FGFR-1 is functional on wild-type sperm and probably signals via the phosphatidylinositol 3-kinase pathway. FGFR-1 activation also resulted in the downstream suppression of mitogen activated protein kinase signaling. These data demonstrate the FGFR-1 is required for quantitatively and qualitatively normal spermatogenesis and has a key role in the regulation of the global tyrosine phosphorylation events associated with sperm capacitation.

Identification of a Novel Testis-Specific Member of the Phosphatidylethanolamine Binding Protein Family, pebp-2

Abstract The phosphatidylethanolamine binding proteins (pebps) are an evolutionarily conserved family of proteins recently implicated in mitogen-activated protein (MAP) kinase pathway regulation, where they are called raf kinase inhibitory proteins. Here, we describe the cloning, cellular localization, and partial characterization of a new member, pebp-2, with potential roles in male fertility. Expression data show that pebp-2 is a testis-specific 21-kDa protein found within late meiotic and haploid germ cells in a stage-specific pattern that is temporally distinct from that of pebp-1. Sequence analyses suggest that pebp-2 forms a distinct subset of the pebp family within mammals. Database analyses revealed the existence of a third subset. Analysis suggests that the specificity/regulation of the distinct pebps subsets is likely to be determined by the amino terminal 40 amino acids or the 3′ untranslated region, where the majority of sequence differences occur. Protein homology modeling suggests that pebp-2 protein is, however, topologically similar to other pebps and composed of Greek key fold motifs, a dominant β-sheet formed from five anti-parallel β strands forming a shallow groove associated with a putative phosphatidylethanolamine binding site. The pebp-2 gene is intronless and data suggest that it is a retrogene derived from pebp-1. Further, pebp-2 colocalizes with members of the MAP kinase pathway in late spermatocytes and spermatids and on the midpiece of epididymal sperm. These data raise the possibility that pebp-2 is a novel participant in the MAP kinase signaling pathway, with a role in spermatogenesis or posttesticular sperm maturation.

Activation of protease-activated receptor-2 leads to inhibition of osteoclast differentiation.

PAR‐2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR‐2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption.

Parthenogenetic activation of rat oocytes and their development (in vitro).

The present study was performed to determine suitable methods for parthenogenetic activation and subsequent development of rat oocytes in vitro. In the first series of experiments, the ability of electrical pulses, strontium, ethanol and ionomycin to activate Sprague-Dawley (SD) rat oocytes was examined. The synergistic effect of strontium and cycloheximide or puromycin was also examined in the second series of experiments. In the third series of experiments, the development of F1 hybrid (SD x Dark Agouti) parthenotes activated with different concentrations of strontium (10-0.08 mM) was compared with that of SD parthenotes. The effect of the timing of activation (10 min and 2, 4 and 6 h after cervical dislocation) was also assessed in a fourth series of experiments. The oocytes activated by strontium showed higher pronuclear formation and cleavage rates than those in the other groups (P < 0.05). Higher blastocyst development was obtained from parthenotes activated by strontium and strontium-cycloheximide compared with the strontium-puromycin group (P < 0.01). However, the total cell number of blastocysts from the strontium-cycloheximide activation group was higher than that of other groups (P < 0.05). With strontium (2.5-10 mM) treatment, 40.9% of blastocysts were obtained from F1 hybrid oocytes, whereas 22.9% were obtained from SD (P < 0.01). The oocytes activated 10 min or 2 h following cervical dislocation showed higher blastocyst development than those of the 4 and 6 h groups (P < 0.01). These results suggest that strontium-cycloheximide produces the highest parthenogenetic activation rate in the rat and that oocytes must be activated by 2 h after cervical dislocation.

Birth of rats following nuclear exchange at the 2-cell stage

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced.

Transposon-mediated generation of targeting vectors for the production of gene knockouts

Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of gene targeting technology include gene replacement (knockins) and conditional vectors which allow for the generation of inducible or tissue-specific gene-targeting events. The assembly of gene-targeting vectors is generally a laborious process requiring considerable technical skill. The procedures presented here report the application of transposons as tools for the construction of targeting vectors. Two mini-Mu transposons were sequentially inserted by in vitro transposition at each side of the region targeted for deletion. One such transposon carries an antibiotic resistance marker suitable for selection in mammalian cells. A deletion is then generated between the two transposons either by LoxP-induced recombination or by restriction digestion followed by ligation. This deletion removes part of both transposons plus the targeted region in between, leaving a transposon carrying the selectable marker flanked by two arms which are homologous to the targeted gene. Targeting vectors constructed using these transposons were electroporated into embryonic stem cells and shown to be effective in gene-targeting events.

The Potential of Rat Inner Cell Mass and Fetal Neural Stem Cells to Generate Chimeras

The rat chimera is an important animal model for the study of complex human diseases. In the present study we evaluated the chimeric potential of rat inner cell masses (ICMs) and fetal neural stem (FNS) cells. In result, three rat chimeras were produced by day 5 (D5) Sprague-Dawley (SD) blastocysts injected with ICMs derived from day 6 (D6) and D5 Dark Agouti (DA) blastocysts；four rat chimeras had been generated by D5 DA blastocyst injected with D5 SD ICMs. For the requirement of gene modification, cultured rat inner cell mass cells were assessed to produce chimeras, but no chimeras were generated from injected embryos. The potential to generate chimeras from rFNS and transfected rFNS cells were tested, but no chimeric pups were produced. Only 2 of 41 fetuses derived from D5 DA blastocyst injection with SD LacZ transfected rFNS cells showed very low number of LacZ positive cells in the section. These results indicate that DA and SD rat ICMs are able to contribute to chimeras, but their potential decreases significantly after culture in vitro (P＜0.05), and rFNS cells only have the potential to contribute to early fetal development.

170 Assessing the potential of stem cells to generate chimeric rats.

Embryonic stem (ES) cells are pluripotent cells derived from inner cell masses (ICMs) of blastocysts. The capacity of pluripotency in differentiation is assumed to contribute to embryonic development to form a chimeric individual when these cells are reintroduced into embryos. Chimeric mice can be routinely generated by aggregation of ES cells with morulae or injection into blastocysts, which are then implanted in pseudopregnant foster mothers. Furthermore, recent studies have demonstrated that bone marrow-derived stem cells and neural stem cells can integrate into the embryonic development in mouse (Geiger et al. 1998 Cell 93, 1055–1065; Clarke et al. 2000 Science 288, 1660–1663). We therefore tried to assess the ability of rat ICMs and neural stem cells to form chimeras by injecting these cells into rat blastocysts. Forty-two rat ICMs from Day 6 blastocysts of Dark Agouti (DA) inbred rat were injected into Day 5 blastocysts of Sprague-Dawleyd (SD) outbred rats; 14 pups were born following embryo transfer of these blastocysts injected into Hooded Wistar (HW) recipients. One male of the 14 pups was coat color-patched and displayed germline transmission. Following embryo transfer of 22 SD blastocysts injected by Day 5 DA ICMs, 7 pups were born and 2 of them were coat color-patched. Nine pups were obtained from 23 DA blastocysts injected by Day 5 SD ICMs; 4 of them were coat color-patched. The ICM cells were isolated and cultured for 6 days. No chimeras were generated by injection of the cultured ICM cells, as assessed by coat color patching. These results suggest that rat embryonic ICMs have potential to develop into chimeras, but the chimeric potential of ICMs was rapidly lost in our culture system. Investigation of potential chimeric development of rat fetal neural stem (rFNS) cells transfected with Lac Z was carried out. Staining was observed in tissues from 2 of 41 E14 fetuses. These results demonstrated that rFNS cells can integrate into the early embryonic environment although the ability of these cells to contribute to chimeric formation was marginal. No coat color chimerism was observed in any of the 88 pups generated from the LacZ-rFNS cell experiments.