Higher magnification images of TIP39 hybridization showed most of the label over the middle third of the epithelium in tubules that were at stage ICVIII, based on the shape of the spermatid nuclei in the inner layers of the epithelium. The protein coding exons of the gene were replaced via homologous recombination by a lacZ/neomycin resistance cassette in F1H4 embryonic stem (ES) cells (a 129-C57BL6 F1 hybrid mouse ES cell line) at Regeneron Pharmaceuticals, Inc. (Tarrytown, NY) using previously established procedures (20) such that the initiating methionine of the lacZ sequence precisely replaces that of TIP39. gene. B, Construct for conditional expression of TIP39 cDNA. Primers ex1F and ex2R produce products of 351 bp from a WT genomic allele, no product from a knock-in allele, and a product of 174 from the transgene used for rescue. C, PCR genotyping of a hybridization histochemistry, mice were perfused with 2.5% XY1 acrolein, 4% paraformaldehyde in PBS. For lacZ histochemistry mice were perfused with 0.2% glutaraldehyde/2% paraformaldehyde in PBS, postfixed for 2 h at 4 C in 0.2% glutaraldehyde cryopreserved in 30% sucrose and sectioned with a cryostat. Paraffin XY1 sections were used for routine histology and immunohistochemistry. Cryostat sections were used for hybridization. The antibody to TIP39 has been previously characterized and described (11). It was used at 1:200 at 4 C overnight and detected using SuperPicture reagent (Invitrogen Corp., Carlsbad, CA) followed by diaminobenzidine. Antibody to GCNA-1 (gift of Dr. George Enders, University of Kansas, Kansas City, KS) was used at 1:50 and detected with Alexa-Fluor 488 conjugated goat antirat IgM (Invitrogen). Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin end labeling of fragmented DNA was performed by Histoserve Inc. (Gaithersburg, MD). 35S-antisense riboprobes for mouse TIP39 and PTH2-R mRNA were produced and used for hybridization as previously described (24,25), and produced the pattern of expression previously established in the brain. For lacZ histochemistry, sections were rinsed in lacZ wash buffer (2 mm MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40 in PBS), and then incubated in the wash buffer containing 5 mm potassium ferricyanide and potassium ferrocyanide and 2 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside) at 37 C for 12C16 h. Image acquisition Bright-field images of tissue sections were obtained with a Zeiss Axioplan 2 microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY), a Zeiss Axiocam HRc, and Zeiss Axiovision software. Combined dark/bright-field and fluorescence images were obtained using an Olympus IX70 microscope (Olympus America, Center Valley, PA) equipped with a Darklite illuminator (Micro Video Instruments, Avon, MA) and a Photometric Coolsnap FX camera (Roper Scientific, Trenton, NJ) using IPLab software (BD Biosciences, Rockville, MD). Images were saved from the acquisition software as tagged image file format files, and contrast and intensity manipulations to entire fields made using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA). Examination of meiotic chromosome spreads The protocol was modified from the procedure described by Moens and Pearlman (26). Testes were dissected, and the capsule was removed. Seminiferous tubules were finely chopped with a scalpel in a petri dish containing 10 ml RPMI 1640 high-glucose media (Invitrogen). The cells were released from the tubules by gentle trituration and filtered through a 40-m cell strainer (BD, Franklin Lakes NJ). The suspension was centrifuged for 8 min Mmp23 at 800 gene contains coding XY1 exons of 206 and a 173 bp. Both coding exons, and the flanked 176-bp intron, were replaced in mouse ES cells via homologous recombination with a cassette encoding -galactosidase and neomycin resistance. Breeding heterozygous mice generated from these ES cells produced homozygous, wild-type (WT), and heterozygous mice of both genders with the expected frequency. Mating 0.05, unpaired two-tailed tests). In this group of mice, seminal vesicle weight was: 0.05, unpaired two-tailed tests). Microscopically, there was a complete absence of spermatids in the testes of adult (ACC) show sections from WT mice and (DCF) show examples of completely synapsed chromosomes, and show synapsed regions in partially synapsed pairs. Evaluation of serum hormones Within the brain TIP39 is synthesized by neurons that project to the hypothalamus (10,11), and TIP39 stimulates GnRH release from hypothalamic slice cultures (12). Because TIP39 mRNA is present in both the testes and brain (15,18), the failure of spermatogenesis in TIP39 knockout (KO) mice could in principle be due to either a change in TIP39 regulated hypothalamic hormones or result from its loss within the testes, or a combination of these. In = 0.01;.