QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Giorgini, F. Articles by Braun, R. E. Search for Related Content PubMed PubMed Citation Articles by Giorgini, F. Articles by Braun, R. E.

Translational repression by MSY4 inhibits spermatid differentiation in mice

Flaviano Giorgini, Holly G. Davies^* and Robert E. Braun

Department of Genome Sciences, University of Washington, Seattle, WA, USA
^* Present address: National Institutes of Health (NIH), National Institute of Diabetes and Digestive Kidney Disorders (NIDDK), Laboratory of Cellular and Developmental Biology (LCDB), 50 South Drive, MSC-8028, Bethesda, MD 20892-8028, USA

View larger version (33K): [in a new window]   Fig. 1. MSY4 gain-of-function transgenes. (A) The upper construct contains the Prm1 5' and 3' UTRs, and a cDNA encoding an HA-tagged version of MSY4. This construct, PMP, is under Prm1-like translational control. The lower construct, PMH, is a variant of the upper transgene and substitutes the hGH 3' UTR for the Prm1 3' UTR. Both constructs are under the control of the Prm1 promoter. (B) Schematic representation of endogenous and transgenic expression of MSY4. The curve labeled MSY4 represents expression of endogenous MSY4 in pachytene spermatocytes and round spermatids. The curve labeled PMH represents transgenic MSY4 expression from the PMH transgene, initiating in round spermatids and continuing in later stage spermatids. The curve labeled PMP represents transgenic MSY4 expression from the PMP transgene initiating in elongated spermatids. Repression and activation of the endogenous Prm1 message during spermatogenesis is represented by the labeled gray boxes. (C) Northern blot analysis of RNA from transgenic PMH founder males and males derived from both the PMH and PMP transgenic lines. Both the endogenous Msy4 mRNA (upper arrow) and the transgenic mRNAs (lower arrows) were detected with a probe prepared from a Msy4 cDNA clone fragment. The endogenous Msy4 mRNA levels serve as an internal loading control.

View larger version (175K): [in a new window]   Fig. 2. Immunocytochemical detection of MSY4 and MSY4-HA. (A,C,E) Testis sections from PMP transgenic mice. (B,D,F) Testis sections from PMH transgenic mice. (A,C) Serial sections of a stage VI tubule from a PMP animal. (B,D) Serial sections of a stage VI tubule from a PMH animal. (A,B) Endogenous MSY4 detected with MSY4 antibody in pachytene spermatocytes (ps) and round spermatids (rs). The transgenic MSY4-HA proteins lack the N-terminal epitope recognized by the MSY4 antibody. (C,D) MSY4-HA detected with HA antibody in elongated spermatids (eds) of PMP and PMH animals, respectively. (E) Detection of MSY4-HA expression in elongated spermatids in a stage VII tubule of a PMP transgenic testis. Notice the absence of MSY4-HA in round spermatids. (F) Expression of MSY4-HA in round and elongated spermatids of a stage VII tubule from a PMH testis. Sections were counterstained with Hematoxylin.

View larger version (145K): [in a new window]   Fig. 3. Histological analysis of testes and cauda epididymides. (A-C) Sections of stage IX tubules from wild-type (A), PMP (B) and PMH (C) mice. (D-F) Enlargements of the boxed areas shown above. As expected, elongating spermatids (egs) are present in stage IX tubules from wild-type and transgenic animals. Elongated spermatids (eds) fail to release in the transgenic epithelium and are eventually engulfed by the Sertoli cells. (G-I) Cauda epididymis from wild-type (G), PMP (H) and PMH (I) mice. The sections were stained with PAS and Hematoxylin.

View larger version (129K): [in a new window]   Fig. 4. Morphology of epididymal sperm. (A) Epididymal sperm from wild-type males. (B) Sperm from a male of the PMP 3464 line. (C) Upper and lower panels show sperm from PMH line 8861 males. Note abnormal head morphology and the extremely small and thin flagella. (D-F) Abnormal sperm from the PMH line 9501.

View larger version (74K): [in a new window]   Fig. 5. Abnormal head morphology of transgenic epididymal sperm. (A) Phase-contrast microscopy of wild-type, PMP and PMH sperm heads. (B) Acridine Orange fluorescence of sperm heads. (i) Wild-type sperm, (ii) wild-type sperm treated with dithiothreitol and 4-vinylpyridine in order to disrupt chromatin integrity, (iii) sperm from the PMP 3464 line, and (iv) sperm from the PMH 9501 line. Sperm heads from PMP and PMH transgenic mice with normal morphology emitted the same green fluorescence as wild-type sperm. Abnormal sperm heads did not fluoresce.

View larger version (47K): [in a new window]   Fig. 6. Analysis of total testis basic proteins from sonication sensitive (SS) and sonication resistant (SR) spermatid nuclei from wild-type and transgenic animals. Total testis basic protein preparations were fractionated by urea-gel PAGE and detected with napthol blue-black staining. Though the total yield of protein was reduced in PMH and PMP basic protein preparations, an equivalent amount of protein was loaded in each lane (15 µg). The left-hand panel contains samples prepared from testis tissue, while the right-hand samples were prepared from epididymal tissue. Wild-type controls are shown in lanes 2, 5, 8 and 11. Lanes 1, 4, 7 and 10 contain protein isolated from PMH mice, while lanes 3, 6, 9 and 12 contain protein isolated from PMP mice. The positions of transition protein 2 (TP2), protamine 1 (PRM1) and mature protamine 2 (PRM2) are labeled. The PRM2 precursor, the partially processed forms of PRM2 and an abnormally processed form of PRM2 are all labeled by PRM2*.

View larger version (61K): [in a new window]   Fig. 7. Electrophoretic mobility shift assay analysis of wild-type and transgenic testis extracts. Lanes 1-3, wild-type testis extracts; lanes 4-9, PMH extracts; and lanes 10-15, PMP extracts. RNA containing the wild-type YRS (wt YRS) was used in lanes 1-8 and lanes 10-14. RNA containing a YRS mutation (C26A) that disrupts MSY4 binding (Giorgini et al., 2001) was used in lanes (9) and (15). Supershifts of the EMSA complex were carried out using either MSY4 antibody (lanes 2, 6, 12), HA antibody (lanes 3, 7, 13) or both (lanes 8 and 14). As a control, pre-immune sera was added to reactions in lanes 5 and 11. No sera was added to reactions in lanes 1, 4 and 10.

View larger version (25K): [in a new window]   Fig. 8. Distribution of endogenous MSY4 and transgenic MSY4-HA in a sucrose gradient. Testis extract was sedimented on a 15% sucrose (w/w) gradient, the absorbance at 254 nm was recorded and 12 fractions collected. Sample 1 is the top of the gradient. Prominent peaks are labeled on the A254 graph. Single ribosome subunits are labeled 40S and 60S, monosomes as 80S/M, disomes as D and trisomes as T. Protein from each fraction was run on a 9% SDS denaturing gel, transferred to nitrocellulose and probed with MSY4 antibody (upper panel) and then HA antibody (lower panel). The location of the 51 kDa standard is indicated on the right.

View larger version (87K): [in a new window]   Fig. 9. Expression analysis of candidate proteins in wild-type and PMH transgenic mice by immunocytochemistry. (A,B) GAPD-S expression in a wild-type (A) and a PMH (B) stage 1 tubule detected with GAPD-S antibody. (C,D) ODF2 expression in a wild-type (C) and a PMH (D) stage 1 tubule detected with ODF2 antibody. (E,F) PRM1 expression in a wild-type (E) and a PMH (F) stage VII tubule detected with HUP1N antibody. (G,H) PRM2 expression in a wild-type (G) and a PMH (H) stage V tubule detected with HUP2B antibody. (I,J) TP1 expression in a wild-type (I) and a PMH (J) stage III tubule detected with TP1 antibody. (K,L) TP2 expression in a wild-type (K) and a PMH (L) stage 1 tubule detected with TP2 antibody. Sections were counterstained with Hematoxylin.