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. 2008 May 15;149(9):4292–4300. doi: 10.1210/en.2008-0419

Tuberoinfundibular Peptide of 39 Residues Is Required for Germ Cell Development

Ted B Usdin 1, Mark Paciga 1, Tim Riordan 1, Jonathan Kuo 1, Alissa Parmelee 1, Galina Petukova 1, R Daniel Camerini-Otero 1, Éva Mezey 1
PMCID: PMC2553379  PMID: 18483145

Abstract

Tuberoinfundibular peptide of 39 residues (TIP39) was identified as a PTH 2 receptor ligand. We report that mice with deletion of Tifp39, the gene encoding TIP39, are sterile. Testes contained Leydig and Sertoli cells and spermatogonia but no spermatids. Labeling chromosome spreads with antibodies to proteins involved in recombination showed that spermatogonia do not complete prophase of meiosis I. Chromosomes were observed at different stages of recombination in single nuclei, a defect not previously described with mutations in genes known to be specifically involved in DNA replication and recombination. TIP39 was previously shown to be expressed in neurons projecting to the hypothalamus and within the testes. LH and FSH were slightly elevated in Tifp39−/− mice, suggesting intact hypothalamic function. We found using in situ hybridization that the genes encoding TIP39 and the PTH 2 receptor are expressed in a stage-specific manner within seminiferous tubules. Using immunohistochemistry and quantitative RT-PCR, TIP39 expression is greatest in mature testes, and appears most abundant in postmeiotic spermatids, but TIP39 protein and mRNA can be detected before any cells have completed meiosis. We used mice that express Cre recombinase under control of a spermatid-specific promoter to express selectively a cDNA encoding TIP39 in the testes of Tifp39−/− mice. Spermatid production and fertility were rescued, demonstrating that the defect in Tifp39−/− mice was due to the loss of TIP39. These results show that TIP39 is essential for germ cell development and suggest that it may act as an autocrine or paracrine agent within the gonads.

GERM CELL development is a highly orchestrated process dependent upon the local milieu. In males a self-renewing population of spermatogonia undergoes mitotic division, and differentiation into spermatocytes. Each generation of spermatocytes progresses in a coordinated wave through two successive meioses to become spermatids, which in turn undergo a highly specialized differentiation to ultimately become spermatozoa. Many genes required for spermatogenesis have been identified, and the steps at which their function is required have been identified by the stages at which spermatogenesis is disrupted when these genes are mutated (1,2,3,4,5). Some are selectively critical for meiosis, which is unique to germ cell development. Most of these gene products are involved in DNA replication, recombination, repair, or other cell autonomous functions.

The classical pituitary hormones FSH and LH, which are critical for multiple processes in gonadal development and function, act through Sertoli and Leydig or granulosa and theca cells, somatic cells in the testes or ovary. Inhibins, activins, and follistatin are produced by somatic cells in the gonads and act on sites outside the gonad. Current evidence indicates that there are also important cellular and paracrine interactions within the gonads at multiple stages of germ cell differentiation (6,7), but relatively few of the critical players have been identified, particularly not any produced by spermatogenic cells (8) or with actions that have been demonstrated to be specific to meiosis.

We purified tuberoinfundibular peptide of 39 residues (TIP39) from bovine hypothalamus on the basis of its selective activation of the PTH 2 receptor (PTH2-R), a family B G protein-coupled receptor (9). In the brain TIP39 is synthesized by two discrete groups of neurons that project to brain areas enriched in PTH2-Rs (10,11). Several experiments suggest that TIP39 acts on PTH2-Rs to modulate hypothalamic, emotional, and nociceptive functions (12,13,14,15). Both TIP39 and the PTH2-R are also expressed in the testes (15,16,17,18), but potential functions in this organ have not been addressed. Many genes are expressed in the testes for which no function within the testes is known, and it is not clear at present whether all such transcripts have physiologically significant roles, or whether “leaky” transcription results in accumulation of mRNA species that are not linked to production of biologically relevant proteins (19). We have now studied mice with deletion of Tifp39, the gene encoding TIP39. Our investigation of these mice shows that expression of Tifp39 is essential for germ cell maturation and that in its absence, spermatocytes fail to complete meiosis.

Materials and Methods

Generation of mutant mice and PCR analysis

The protein coding exons of the Tifp39 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. Tifp39+/− ES cells were injected into mouse C57Bl6 blastocysts to produce Tifp39+/− founders. A mouse TIP39 cDNA was subcloned into the BamH1 and XhoI sites of the vector pCCALL2 (21) (a gift of Dr. Corrine Lobe, University of Toronto, Toronto, Ontario, Canada), and C57Bl6 mouse embryos were injected with the pCALL2_TIP39 construct to produce TgFlxStpTIP39cDNA founder mice. Embryo manipulations were performed by the National Institute of Mental Health transgenic core facility. TgPrm1Cre mice, originally produced by O’Gorman et al. (22), were obtained from Jackson Laboratories (Bar Harbor, ME). ROSA26-eYFP mice (23) were a gift of Dr. Frank Costantini (Columbia University, New York, NY). Mice were genotyped by transgene-specific PCR of tail biopsy DNA using primers as diagrammed (see Fig. 7) (Ex1F 5′-GTGTTGCCCTGCCCCTCG-3’, Ex2R 5′-TGTAAGAGTCCAGCCAGCGG-3’, GenF 5′-TTCTGGGCGGGATGATGAC-3’, GenR 5′-TCGGGATTTCGGGGCGTTA-3’, and NeoF 5′-GCTTCCTCGTGCTTTACGGTATC-3’) and for the protamine-Cre transgene (protCre_F1 5′-GGTCCTGGTCCTCTTTGACTTCA-3’ and protCre_R1 5′-TGCGAACCTCATCACTCGTTGC-3’). All animal procedures were performed in accordance with National Institutes of Health guidelines.

Figure 7.

Figure 7

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Rescue of TIP39 null mouse. A, Tifp39−/− construct and genotyping. Primers GenF and GenR flank the two exons of TIP39 and produce a band of 1300 bp from a WT allele; a much larger band would be produced from a knock-in allele, but it is not seen under the conditions used. Primers NeoF and GenR produce a band of 600 bp from a KO allele. B indicates Bgl I sites in the Tifp39 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 Tifp39−/− mouse containing the Cre-recombinase activated TIP39 transgene and the protamine Cre transgene (left), and of a Tifp39+/− negative for the Cre-recombinase activated TIP39 transgene (right). Sections from testes from a 6-wk-old Tifp39−/−, TgFlxStpTIP39cDNA, TgPrt1Cre (D), a Tifp39+/− mouse (E), and a Tifp39−/− mouse (F) are shown. Mice were perfusion fixed with buffered 4% paraformaldehyde. Scale bars, C–E 50 μm.

Tissue histology

For routine histology testes were fixed by immersion in 4% paraformaldehyde in PBS or Bouin’s fixative. For immunohistochemistry mice were perfused with Bouin’s fixative. For in situ hybridization histochemistry, mice were perfused with 2.5% 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 sections were used for routine histology and immunohistochemistry. Cryostat sections were used for in situ 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 in situ 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 12–16 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-μm cell strainer (BD, Franklin Lakes NJ). The suspension was centrifuged for 8 min at 800 × g, and the cells resuspended in 10 ml RPMI 1640 and pelleted again. The resulting pellet was resuspended in 1 ml 0.5% NaCl after any residual media had been removed. Fifteen microliters of the cell suspension were added to glass slides with hydrophobic rings (BD), and the cells were allowed to adhere without drying out for 10–15 min. The slides were fixed in 2% paraformaldehyde and 0.03% sodium dodecyl sulfate for 3 min, 2% paraformaldehyde for 3 min, washed three times in 0.4% Photo-Flo 200 (Kodak, Rochester NY) for 1 min, air dried, and stored at −20 C. For immunostaining the slides were incubated with blocking solution (1% donkey serum, 0.3% BSA, and 0.05% Triton X-100 in PBS) for 20 min at 37 C in a humidity chamber. Primary antibodies, anti-Scp3 mouse polyclonal raised against a glutathione S-transferase fusion of the full-length rat Scp3 protein (27), rabbit anti-Scp1 antibody [gift from C. Hoog (Karolinska Institute, Stockholm, Sweden) (28)], Dmc1 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and H2AX antibodies (Trevigen, Gaithersburg, MD), were diluted in blocking buffer and incubated under the same conditions for 1–2 h. After two 5-min washes in 0.4% Photo-Flo/PBS solution, slides were blocked for an additional 5 min and incubated with fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 20 min at 37 C. The slides were washed twice with 0.4% Photo-Flo in PBS, rinsed twice with 0.4% Photo-Flo, and allowed to air-dry. Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) was added, and the slides were coverslipped and viewed with a Leica DM5500B fluorescent microscope (Leica Microsystems, Bannockbur, IL). The images were captured at 1000× magnification with OpenLab software (Improvision, Waltham, MA) and processed using Adobe Photoshop.

Hormone determinations

Hormone measurements were performed by the University of Virginia Center for Research in Reproduction, Ligand Assay and Analysis Core Laboratory on serum collected from trunk blood of 60- to 70-d-old mice. Sensitivities, and intraassay and interassay variabilities are available on the laboratory web site (http://www.healthsystem.virginia.edu/internet/crr/CVDATA_2007.pdf).

Quantitative RT-PCR (Q-RT-PCR)

RNA was isolated from testes stored in RNALater (Ambion, Inc., Austin, TX), by homogenization in TRIzol (Invitrogen) in individual ceramic bead-containing microcentrifuge tubes (Lysing Matrix D; Qbiogene, Inc., Irvine, CA) using a Fastprep system (Qbiogene). After deoxyribonuclease I treatment, cDNA was prepared using an oligo deoxythymidine primer and ThermoScript Reverse Transcriptase (Invitrogen), treated with ribonuclease H, and purified with a G-50 micro-spin column. cDNA was amplified using primers TIP39q3 5′-AGGTGATGGAGACCTGCCAGATG and TIP39q4 5′-CTTCTGCATGTAAGAGTCCAGCCA, mP2Rq1 5′-GTTTGCCTTCTTTGTGCTCCC and mP2Rq2 5′-TGACATTTTCCCCACTGTTCCTC, or mGAPDH_U1 5′-CAAAAGGGTCATCATCTCCGC and mGAPH_L1 5′-ACACATTGGGGGTAGGAACACG in 25 μl reactions containing 0.05 μl KlentaqLA enzyme (Wayne Barnes, Washington University, St. Louis, MO) and 1 × EvaGreen (Biotium, Inc., Hayward, CA) in a MJ DNA Engine Opticon 2 System (Bio-Rad Laboratories, Inc., Hercules, CA), using one cycle of 93 C for 2 min, 40 cycles of 93 C for 15 sec, 50.7 C (for TIP39) or 60.7 C (for PTH2-R) for 30 sec, 68 C for 35 sec, with an optical read during an 85 C for 10-sec step. Single products were confirmed from melting curves and analysis of final products on agarose gels. A comparative threshold (CT) was obtained in each experiment by selecting a fluorescence intensity on the linear range for all samples amplified with a particular primer pair in that experiment. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified from reactions set up in parallel and amplified using conditions optimized for the primary target. Relative expression was calculated as 2−DDCT, where DCT = CT (TIP39 or PTH2-R) − CT (GAPDH) and DDCT = DCT − baseline DCT, with baseline DCT set arbitrarily as the smallest value in a given experiment.

Southern blots of reporter transgenic testes

DNA was isolated from testes using a Puregene DNA purification kit (Gentra Systems, Minneapolis, MN) according to the manufacturer’s instructions, with homogenizations performed in individual disposable ceramic bead-containing tubes. DNA (100 ng/reaction) was amplified (94 C for 15 sec, 68 C for 4 min, 35 cycles) with primers ROSA 5′F (5′-GCAATACCTTTCTGGGAGTTCTCTG) and YFP-R (5′-GAAGTTCACCTTGATGCCGTTC). These primers are predicted to amplify a 3.5-kb product from the unmodified ROSA26-eYFP transgene and a 847-bp product from the transgene after Cre-recombinase catalyzed recombination. After agarose gel electrophoresis, PCRs were transferred to a nitrocellulose membrane, hybridized with a 32P-labeled probe generated from the recombined 847-bp ROSA-5′F/YFP-R PCR product, and exposed to a phosphor imager plate for 24 h after a high-stringency wash.

Results

Generation of TIP39 null mice

The mouse Tifp39 gene contains coding 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 β-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 Tifp39−/− mice of either gender with WT mice produced no pups.

Phenotypical description

On external examination of Tifp39−/− mice, there was no obvious effect of TIP39 deletion. Macroscopically, testes from Tifp39−/− males were obviously smaller than those of WT or heterozygous animals (Fig. 1). From a group of mice analyzed at postnatal d 80–90, Tifp39+/+ testis length (cm) was 0.86 ± 0.014 (n = 22) and Tifp39−/− 0.475 ± 0.018 (n = 13) (mean ± sem). The testes from Tifp39−/− weighed much less than those from WT mice, and the seminal vesicles were slightly lighter. From a separate group of Tifp39+/+ and Tifp39−/− mice analyzed at postnatal d 70–80, individual testis weight (mg) was: Tifp39+/+, 0.111 ± 0.002 (n = 12, six mice); Tifp39−/−, 0.025 ± 0.007 (n = 10, five mice) (mean ± sem; P < 0.05, unpaired two-tailed t tests). In this group of mice, seminal vesicle weight was: Tifp39+/+, 0.26 ± 0.01 (n = 5); Tifp39−/−, 0.21 ± 0.003 (n = 5) (mean ± sem; P < 0.05, unpaired two-tailed t tests). Microscopically, there was a complete absence of spermatids in the testes of adult Tifp39−/− mice (Fig. 2 and supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). We saw no difference between WT and Tifp39−/− testes at postnatal d 7. By postnatal d 14, a few more tubules with pyknotic cells were seen in the testes from Tifp39−/− mice than in WT testes. The difference was clear by postnatal d 21 when round spermatids could be seen in some tubules from WT mice, whereas tubules in testes from Tifp39−/− animals contained no spermatids, and many contained pyknotic nuclei (Fig. 3). Thus, the difference between Tifp39−/− and WT mouse testes appeared before the completion of the first meiotic prophase in postnatal testes, and no postmeiotic germ cells were apparent in testes from adult Tifp39−/− mice. The germ cell-specific antibody GCNA1 strongly labels nuclei of spermatogonia and early spermatocytes, and labels late stages of spermatocyte development much more weakly (29). GCNA1 labeling showed a similar population of spermatogenic cells in WT and Tifp39−/− testes at postnatal d 14. At postnatal d 21, strong labeling persisted in WT mice in a subpopulation of cells in the basal layer of the seminiferous epithelium, as previously described (29). In contrast, the majority of cells in Tifp39−/− mice had strong GCNA1 labeling at postnatal d 21, indicating the persistence of early spermatocytes and spermatogonia, and the lack of more mature spermatocytes or spermatids (Fig. 4). Leydig and Sertoli cells could be identified in the Tifp39−/− testes based on morphology and location, and labeling of Sertoli cells with vimentin (data not shown). Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5’-triphosphate-biotin end labeling of fragmented DNA indicated widespread apoptotic cell death in the testes of adult Tifp39−/− mice (data not shown). Thus, in the absence of TIP39, there was a failure of spermatocytes to progress through meiosis.

Figure 1.

Figure 1

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Testes from Tifp39−/− mice are small. Testes from 6-wk-old Tifp39−/− mice (B) are obviously smaller than testes from a littermate WT mouse (A).

Figure 2.

Figure 2

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Testes from Tifp39−/− mice lack mature spermatogenic cells. Comparison of testes from 6-month-old littermate WT (A and B) and Tifp39−/− (C and D) mice show that seminiferous tubules from Tifp39−/− mice have far fewer cells and a much more limited set of cells than WT mice. Note the lack of cells with condensed chromosomes and the absence of round spermatids in the Tifp39−/− testes. Sertoli cell and pyknotic nuclei can be seen in the Tifp39−/− testes. Testes immersion fixed in 10% formalin were stained with hematoxylin and eosin. See supplemental Fig. 1 for a higher quality image of testes from 60-d-old mice. Scale bars, A and C 100 μm, and B and D, 10 μm.

Figure 3.

Figure 3

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Postnatal onset of spermatogenesis defect in Tifp39−/− mice. Top panels (A–C) show sections from WT mice and bottom panels (D–F) Tifp39−/− littermates at postnatal d 7 (A and D), 14 (B and E), and 21 (C and F). Some tubules containing pyknotic nuclei can be seen in the Tifp39−/− mouse at postnatal d 14. On postnatal d 21, round spermatids can be seen in the WT but not the Tifp39−/− mouse, and many more degenerating cells are apparent in the Tifp39−/− testes. Testes immersion fixed in buffered 4% formaldehyde were stained with hematoxylin. Scale bar, 50 μm.

Figure 4.

Figure 4

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Spermatogenic cells persist in Tifp39−/− testes. Testes from WT (A and B) and Tifp39−/− mice (C and D) were labeled with antibody GCNA1 at postnatal d 14 (A and C) and 21 (B and D). Testes were immersion fixed in Bouin’s fixative. Scale bar, 50 μm.

Characterization of meiotic arrest

During prophase of the first meiotic division, homologous chromosomes (homologs) synapse, and recombination takes place, followed by separation of the homologs to daughter nuclei. Immunolabeling of chromosome spreads demonstrated that homologous synapsis is incomplete in the spermatocytes of Tifp39−/−− mice, with only about half of the chromosomes showing homologous alignment (Fig. 5). Because chromosome synapsis is intimately interlinked with homologous recombination and synapsis failure is often the result of a recombination defect, we used marker antibodies to assess the progression of recombination in the Tifp39−/− spermatocytes. Recombination is normally initiated by the formation of double-stranded DNA breaks, followed by rapid phosphorylation of the histone variant H2AX in the vicinity of the break. As recombination progresses, the key DNA repair enzymes Rad51 and Dmc1 localize to the ends of the breaks and perform the search for homologous chromosome that eventually leads to side-by-side alignment (synapsis). After the DNA breaks have been repaired, phosphorylated H2AX is no longer detected on the chromosomes, and Rad51 and Dmc1 complexes disassemble. In Tifp39−/− spermatocytes, recombination was properly initiated as demonstrated by H2AX phosphorylation (Fig. 5). The loading of Rad51 and Dmc1 was also not affected (Fig. 5; data not shown), and normal break repair was indicated by the disappearance of phospho-H2AX staining and Dmc1 foci from the synapsed chromosomal regions. Surprisingly, despite this apparently normal recombination and synapsis progression in some chromosomes, others either only partially repaired their breaks (and were partially aligned) or failed to repair breaks at all and remained completely unsynapsed. This was a rather unusual finding because at the time of meiotic arrest in previously characterized recombination/synapsis mutants, the alignment state of the chromosomes in a given nucleus is uniform. Either no homologous synapsis is present [i.e. Spo11−/−, Dmc1−/−, Msh4−/−, Msh5−/−, and Hop2−/− mice (27,30,31,32,33,34,35)], or, if a defect occurs at later stages, all chromosomes are homologously synapsed [i.e. Mlh1−/− mice (36)]. This difference from previously characterized meiosis defects suggests that the defect in Tifp39−/− mice may not be within an intrinsic component of the recombination apparatus but may result from loss of a signal involved in progression of the meiotic program or its coordination with spermatogenesis.

Figure 5.

Figure 5

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Meiotic arrest of Tifp39−/− spermatocytes. A, Chromosome spreads from Tifp39−/− spermatocytes were coimmunostained with anti-Scp3 antibodies (green in all images) to show chromosomal cores and anti-Scp1 antibodies (red) to indicate the synapsed regions. Merge of green and red staining gives yellow color. B and C, Immunostaining for the phosphorylated form of histone H2AX (gH2AX; red in B) and Dmc1 (red in C) protein. Note the lack of the staining at the synapsed regions where the repair of double-stranded breaks is completed. Arrows show examples of completely synapsed chromosomes, and arrowheads 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 Tifp39−/− males, serum FSH was slightly elevated, and LH and testosterone did not significantly differ between the genotypes, suggesting that hypothalamo-pituitary dysfunction is unlikely to account for the gonadal phenotype of these mice and that Leydig cell function is intact [FSH (ng/ml): WT 27.5 ± 2.4 (n = 5) and KO 38.1 ± 2.0 (n = 5), P = 0.01; LH (ng/ml): WT 0.51 ± 0.27 (n = 7) and KO 0.76 ± 0.39 (n = 10), P = 0.63; testosterone (ng/dl): WT 99 ± 24 (n = 13) and KO 179 ± 59 (n = 14), P = 0.23; mean ± sem, unpaired two-tailed t tests].

Testicular mRNA expression and protein synthesis

In situ hybridization showed that TIP39 mRNA was expressed within some but not all tubules in WT mice (Fig. 6), indicating that it is expressed in a stage-specific manner. Hybridization with the TIP39 probe also confirmed the absence of TIP39 mRNA in Tifp39−/− mice (data not shown). The tissue processing used for in situ hybridization makes it difficult to identify the developmental stage of spermatogenic cells. Higher magnification images of TIP39 in situ hybridization showed most of the label over the middle third of the epithelium in tubules that were at stage I–VIII, based on the shape of the spermatid nuclei in the inner layers of the epithelium. In adult mice labeling with an antibody to TIP39 protein was most clearly detected in mature spermatids. However, labeling of testes from mice at postnatal d 14 showed that TIP39 was also synthesized by cells in the seminiferous epithelium at earlier stages of development. The lacZ sequence replaces the TIP39 sequence in the construct used to produce Tifp39 mutant mice, so β-galactosidase expression in Tifp39+/− and Tifp39−/− mice should reflect the activity of the TIP39 promoter. β-Galactosidase histochemistry in Tifp39+/− mice shows labeling consistent with the antibody and in situ hybridization labeling (supplemental Fig. 2). There is also strong labeling of interstitial cells due to an endogenous enzyme activity that can be seen in WT mice, which limits the ability to interpret the β-galactosidase histochemistry. In Tifp39−/− mice β-galactosidase labeling is almost completely absent from the seminiferous tubules.

Figure 6.

Figure 6

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TIP39 and PTH2-R expression in WT testes. Using in situ hybridization histochemistry, TIP39 mRNA is found in the seminiferous epithelium of adult WT testes. A, When viewed under a mixture of transmitted and reflected (dark-field) illumination, it is apparent that TIP39 mRNA, indicated by bright silver grains in this emulsion-dipped autoradiogram, is present in some but not all tubules. B, At higher magnification, under bright-field illumination in which silver grains appear black, the labeling can be seen in the middle part of the epithelium of a labeled tubule. Antibody labeling shows TIP39-like immunoreactivity most clearly toward the center of seminiferous tubules from adult mice (perfused with Bouin’s fixative) (C) and throughout the epithelium at postnatal d 14 (immersion fixed with Bouin’s fixative) (D). E and F, In situ hybridization also shows PTH2-R mRNA in some tubules of adult testes. Scale bars, A and E 100 μm, B and F 20 μm, and C and D 50 μm.

TIP39 was identified as a selective ligand for the PTH2-R. Expression of the PTH2-R has previously been described in rat testes (16). Using in situ hybridization, we confirmed that its mRNA is present in mouse testes and observed that, like TIP39, it is expressed with a stage-specific pattern in seminiferous tubules (Fig. 6, E and F). Both TIP39 and PTH2-R mRNA were clearly detected by PCR of reverse-transcribed testes RNA (Q-RT-PCR) at postnatal d 21, and rapidly increased after this age (Table 1). Q-RT-PCR from the entire organ is less sensitive than visualization of individual cells by in situ hybridization or immunolabeling. The RT-PCR confirms that TIP39 is expressed before the completion of meiosis and that the PTH2-R has a similar expression pattern. Using both in situ hybridization and RT-PCR, we found that PTH2-R mRNA was not detected in the testes of adult Tifp39−/− mice. PTH2-R mRNA was present in the brain of Tifp39−/− mice.

Table 1.

TIP39 and PTH2-R Q-RT-PCR

Postnatal d Relative P2R expression Relative TIP39 expression No.
12 20 ± 8 27 ± 5 3
16 13 ± 4 11 ± 4 3
17 22 ± 6 22 ± 10 3
21 518 ± 720 912 ± 701 4
45 1,336 ± 271 13,009 ± 9,360 5
120 9,361 ± 2,993 65,619 ± 46,269 4
KO 60 5 ± 1.4 2 ± 0.4 4
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Values are mean ± sem. P2R, PTH2-R. 

Rescue by transgenic expression of TIP39 cDNA

The relatively normal serum levels of FSH and LH and the synthesis of TIP39 within the testes suggest that the loss of gonadal TIP39 is responsible for the defect in spermatogenesis. However, defects in the pattern of pituitary hormone secretion could potentially be responsible. It is also possible that replacement of the Tifp39 gene with the lacZ/neomycin cassette affects the function of a different gene. We tested the hypothesis that the loss of TIP39 signaling within the testes is responsible for the failure of sperm cell development by performing a rescue experiment. First, we created transgenic mice containing a construct from which TIP39’s cDNA sequence is expressed after Cre-recombinase mediated excision of an upstream sequence containing a stop signal. These “floxed-stop TIP39 cDNA” transgenic mice (TgFlxStpTIP39cDNA) were bred with Tifp39+/− mice. Mice from a second transgenic line, that incorporates a protamine-1 promoter driven Cre-recombinase transgene (TgPrm1Cre), were also bred with Tifp39+/− mice. Protamine is exclusively expressed in the testes, and testis-specific expression of Cre recombinase by TgPrm1Cre mice has been described (22).

Matings were subsequently performed between the Tifp39+/−, TgFlxStpTIP39cDNA, and Tifp39+/−, TgPrm1Cre mice. From eight such matings, a total of 190 pups were analyzed, and six male mice were identified that were homozygous null at the Tifp39 locus and contained both the “floxed-stop TIP39 cDNA” and the protamine-1 promoter driven Cre-recombinase transgenes (Tifp39−/−, TgFlxStpTIP39cDNA, TgPrm1Cre). Testes from Tifp39−/−, TgFlxStpTIP39cDNA, TgPrm1Cre mice contained spermatogenic cells at all stages of differentiation, and at postnatal d 60 could not be distinguished from WT testes. In contrast, testes from Tifp39−/− mice transgenic for either the “FlxStpTIP39cDNA” transgene or the protamine-1 Cre-recombinase transgene, but not both, were not distinguishable from those of Tifp39−/− mice (Fig. 7). Mating a Tifp39−/−, TgFlxStpTIP39cDNA, and TgPrm1Cre male with a WT female led to viable pups, demonstrating return of fertility. Female Tifp39−/− mice are also sterile and have very small atretic ovaries without follicles (Fig. 8). The ovaries from Tifp39−/−, TgFlxStpTIP39cDNA, TgPrt1Cre female mice were atretic and not distinguishable from those of Tifp39−/−mice.

Figure 8.

Figure 8

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Ovaries from Tifp39−/− mice are atretic. An ovary from a 6-wk-old WT mouse (A) contains follicles at several stages, whereas an ovary from a Tifp39−/− littermate (B) is small and contains no follicles.

Protamine is a basic protein that replaces histones in postmeiotic spermatids. We examined the developmental stage in which Cre recombinase becomes functionally active in the testes of TgPrt1Cre mice by performing PCR on DNA from the testes of mice that contain a reporter transgene and the protamine-1 Cre-recombinase transgene using primers flanking the “floxed” sequence in the reporter transgene. The PCR product produced from the recombined DNA was not detectable in DNA from postnatal d 19 or earlier mice. It could be detected on Southern blots at postnatal d 25, and was robustly detected at postnatal d 32 and beyond (Fig. 9).

Figure 9.

Figure 9

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Cre-recombinase function in TgPrt1Cre testes. DNA prepared from the testes of pups harvested at various ages from a cross between a TgPrt1Cre and a homozygous ROSA26-eYFP mouse was amplified using primers flanking the two lox P sites in the ROSA26-eYFP transgene. An 847-bp product is formed after recombination. A Southern blot of the PCR products was hybridized with a 32P-labeled probe made from this 847-bp recombined PCR product. Mice were separately genotyped for the protamine-Cre transgene. Age (postnatal days) and protamine-Cre status are indicated in each lane.

Discussion

Expression of the Tifp39 gene is essential for the development of mature germ cells in both males and females. The age at which cellular differences between the testes of WT and KO mice become apparent, the histology of the testes from KO mice, and the pattern of labeling with antibodies to proteins involved in chromosomal recombination suggest that in males there is a block during prophase of meiosis I, with subsequent death of developing spermatocytes. We showed that TIP39 is normally expressed by developing spermatogenic cells and that introduction of a TIP39 cDNA sequence into Tifp39−/− mice under control of the protamine1 promoter restores spermatid production and fertility to Tifp39−/− males, so we infer that TIP39 production by spermatogenic cells is required for progression through meiosis. We have not examined Tifp39−/− females beyond the observation that they lack ovarian follicles, but we suggest that TIP39 is likely to have a similar role in oocyte development. TIP39 activates the PTH2-R, and PTH2-R mRNA is expressed by spermatogenic cells, so it is likely that TIP39 acts via the PTH2-R in the gonads.

Many, presumably local, mediators that are known to act as intercellular signals in other organs are produced in the testes (37). In general, their physiological significance is not clear. For instance, fibroblast growth factors and IGFs and their receptors are present in the testes, and have been proposed to be paracrine factors, with most reports suggesting effects at the premeiotic stage of spermatogonia, or in the function of Leydig or Sertoli cells (reviewed in Ref. 7). Both redundancy and effects in other organs may contribute to the difficulty in identifying their specific roles in the testes. The requirement of TIP39 for progression through meiosis may be apparent both because there is no other factor that can replace it and because it does not have essential functions in other organs.

Because spermatocytes do not complete meiosis in the absence of TIP39, and there is expression of TIP39 by spermatogenic cells before any cells have completed meiosis, the simplest explanation for the site of action of TIP39 is that it acts on spermatocytes before or during meiosis. Thus, it may be an autocrine factor. Action as a germ cell produced autocrine agent is also consistent with the requirement of TIP39 for female gonad development, although its synthesis has not been examined in female gonads. Rescue by the protamine-1 promoter driven construct is then paradoxical because the promoter is thought to become active in postmeiotic cells. A likely explanation is that there is some production of Cre recombinase within the testes of TgPrm1Cre mice before or early in meiosis. We attempted to identify the cells in which Cre recombinase is active by immunolabeling yellow fluorescent protein (YFP) in mice containing the protamine-Cre transgene and a reporter construct that expresses YFP after Cre-recombinase mediated excision of an upstream stop signal. We were unable to detect any YFP expression histologically in the testes of these mice, presumably because by the time significant amounts of Cre recombinase has been synthesized, transcription or translation is repressed. We were able to show that there was a development related increase in Cre-recombinase expression by assaying recombination of the DNA sequence in the reporter construct. However, the first age at which we detected recombination is well after the start of meiosis, as previously described for a similar construct (38). We think that there is likely to be a low level of recombinase expression earlier, which these assays do not detect.

TIP39 antibody labeling and β-galactosidase histochemistry, including the almost complete disappearance of histochemical product from tubules of Tifp39−/− mice, strongly suggest that the vast majority of TIP39 expression in testes is by spermatogenic cells. A very small amount of histochemical product in tubules of Tifp39−/− mice is likely to reflect synthesis by spermatogenic cells before the stage of meiosis in which they die. However, the possibility of a low level of expression by Sertoli cells cannot be completely excluded.

Alternative explanations for both spermatocyte death in Tifp39−/− mice during meiosis and rescue in these mice expressing a TIP39 cDNA under protamine-1 promoter control are possible. For instance, another potential explanation for the rescue is that a small number of spermatocytes may “slip through” meiosis in Tifp39−/− mice. These postmeiotic cells would then produce ample TIP39 in the Tifp39−/−, TgFlxStpTIP39cDNA, TgPrm1Cre mice. TIP39 released from these postmeiotic cells would diffuse throughout the seminiferous epithelium, act on cells entering meiosis, and facilitate the passage of more cells through meiosis, and, thus, ultimately rescue the population. Ultimately, in vitro systems that are not currently well developed may be required to determine the mechanism of action of TIP39 in the gonads.

There are several steps during spermatogenesis at which TIP39 could act directly. MAPK pathways have been reported to be involved in meiotic progression, but the responsible ligand(s) are not known (8). Ligand activation of G protein-coupled receptors can lead to MAPK activation, and TIP39 activates the G protein-coupled PTH2-R, so it is possible that TIP39 affects meiosis through a MAPK pathway. Activation of the PTH2-R stimulates cAMP accumulation in transfected cells, and critical roles for cAMP in the maturation of germ cells are well documented at late stages of spermatogenesis (spermiogenesis) (39). Like other family B G protein-coupled receptors, activation of the PTH2-R also leads to an increase in intracellular Ca2+ in some cells (40), so cAMP independent functions of the PTH2-R could be critical for its function in the testes. Interestingly, several DNA recombination factors have recently been shown to be regulated by Ca2+ (41,42). The particular pattern of failure of meiosis in the Tifp39−/− mice has not previously been described. The nonuniformity of the state of recombination and repair of chromosomes suggests that a novel mechanism is responsible. However, it is also possible that the apparent novelty reflects the limited number of studies using current immunolabeling techniques that provide a detailed picture of events during meiosis. The striking feature of chromosome labeling in these mice is that some chromosomes seem to synapse appropriately, whereas others are observed at a variety of stages. This suggests that TIP39 may be required for production of a common limiting factor.

The inference that TIP39 action on the PTH2-R in the testes is required for germ cell maturation is based on TIP39’s ability to activate the PTH2-R and the expression of PTH2-R mRNA in the testes. However, an essential role of the PTH2-R has not yet been demonstrated. PTH2-R mRNA was not detected in Tifp39−/− mice, which may reflect the absence of spermatids. The measurements of PTH2-R mRNA may not be sensitive enough to determine whether there is expression by the spermatogenic cells at early developmental stages that persist in the Tifp39−/− mice.

RT-PCR (our unpublished data) shows that PTH2-R and TIP39 mRNA are present in nonhuman primate testes. Thus, TIP39 and the PTH2-R may have similar roles in human germ cell maturation to that proposed for the mouse. Unlike rodent PTH2-Rs, the human PTH2-R is activated by PTH in addition to TIP39 (40). This brings up the possibility that inactive fragments of PTH such as those lacking amino-terminal residues, which are produced in diseases such as chronic/end-stage renal disease (43), could contribute to the infertility (44) associated with these diseases by blocking testicular PTH2-Rs. However, because of the difference in ligand recognition specificity of rodent and human PTH2-Rs, and the multiple endocrine abnormalities often present in these patients, it will be difficult to test this idea.

Our data show that expression of the Tifp39 gene is essential for the formation of mature germ cells. It is one of relatively few factors selectively critical for germ cell development in both males and females, and is unique in having no obvious association with DNA replication. TIP39 produced from the Tifp39 gene is a neuromodulator in the brain and may function as a secreted autocrine or paracrine factor in the testes. Based on the expression of both TIP39 and the PTH2-R by spermatogenic cells, we hypothesize that TIP39 signals between spermatogenic cells and is responsible for a previously unknown action within seminiferous tubules. The causes of most cases of infertility are unknown, and it is possible that defects in TIP39 signaling are among them. Whether manipulation of TIP39 signaling could be useful for control of fertility may also be considered.

Supplementary Material

[Supplemental Data]
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Acknowledgments

We thank Mark Sleeman, David Valenzuela, Andrew Murphy, and George Yancoupolos of Regeneron Pharmaceuticals, Inc. (Tarrytown, NY) for the development of the tuberoinfundibular peptide of 39 residues mutant embryonic stem cell lines, Jim Pickel of the National Institute of Mental Health Intramural transgenic core facility for mouse development, Shuaike Ma for performing in situ hybridization experiments, and Millan Rusnak for labeling tissue. We appreciate helpful comments on the manuscript from Mary Ann Handel and Howard Nash.

Footnotes

This research was supported by the Intramural Program of the National Institutes of Health, National Institute of Mental Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 15, 2008

Abbreviations: CT, Comparative threshold; ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; PTH2-R, PTH 2 receptor; Q-RT-PCR, quantitative RT-PCR; TIP39, tuberoinfundibular peptide of 39 residues; WT, wild type; YFP, yellow fluorescent protein.

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Supplementary Materials

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