Transcription and post-transcriptional regulation of spermatogenesis

Spermatogenesis in mammals is achieved by multiple players that pursue a common goal of generating mature spermatozoa. The developmental processes acting on male germ cells that culminate in the production of the functional spermatozoa are regulated at both the transcription and post-transcriptional levels. This review addresses recent progress towards understanding such regulatory mechanisms and identifies future challenges to be addressed in this field. We focus on transcription factors, chromatin-associated factors and RNA-binding proteins necessary for spermatogenesis and/or sperm maturation. Understanding the molecular mechanisms that govern spermatogenesis has enormous implications for new contraceptive approaches and treatments for infertility

shRNA Off-Target Effects <i>In Vivo</i>: Impaired Endogenous siRNA Expression and Spermatogenic Defects

<div><p>RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related <u>r</u>eproductive <u>h</u>omeob<u>ox</u> 3 (<i>Rhox3</i>) gene paralogs. Using a <i>Rhox3</i> short hairpin (sh) RNA with 100% complementarity to all 8 <i>Rhox3</i> paralogs, expressed from a CRE-regulated transgene, we successfully knocked down <i>Rhox3</i> expression in male germ cells <i>in vivo</i>. These <i>Rhox3</i>-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced <i>Rhox3</i> expression, we generated mice expressing the <i>Rhox3</i>-shRNA but lacking the intended target of the shRNA—<i>Rhox3</i>. These double-mutant mice had a phenotype indistinguishable from <i>Rhox3</i>-shRNA-expressing mice that was different from mice lacking the <i>Rhox3</i> paralogs, indicating that the <i>Rhox3</i> shRNA disrupts spermatogenesis independently of <i>Rhox3</i>. <i>Rhox3</i>-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the <i>Rhox3</i> shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions <i>in vivo</i>.</p></div

<i>Rhox3</i>-shRNA mice have reduced expression of endo-siRNAs but not miRNAs.

<p>(A) Relative miRNA levels in testes of 15 days-old control and <i>Rhox3</i>-shRNA mice. (B) Relative endo-siRNA levels in testes of 15 days old control and <i>Rhox3</i>-shRNA mice. Values denote the mean fold change ±S.E. <i>Rhox3</i>-shRNA mice, <i>Rhox3</i>-shRNA;<i>Stra8</i>-iCre double-transgenic mice; Control mice, <i>Rhox3</i>-shRNA single-transgenic mice.</p

Primers used for qRT-PCR Analysis.

<p>Primers used for qRT-PCR Analysis.</p

Histological analysis of <i>Rhox3</i>-shRNA mice.

<p>(A–F) Images of periodic acid Schiff (PAS)-hematoxylin stained cross-sections of adult testis from <i>Rhox3</i>-shRNA mice (Line 2). Roman numbers indicate seminiferous tubule stages. (A) Arrows, apoptotic pachytene spermatocytes. (B) Brackets, missing generations of pachytene spermatocytes and round spermatids. (C) Arrows, apoptotic metaphase spermatocytes. (D) Double arrows, apoptotic pachytene spermatocytes; arrow, a clump of apoptotic cells; arrow-heads, apoptotic round spermatids. (E) Arrow, a clump of spermatids, which are so-called “giant cells” that will probably undergo apoptosis. (F) Arrows, abnormal elongating spermatids; arrow heads, elongating spermatids abnormally deep in the epithelium. Scale bar: 25 μm. <i>Rhox3</i>-shRNA mice, <i>Rhox3</i>-shRNA;<i>Stra8</i>-iCre double-transgenic mice; Control mice, <i>Rhox3</i>-shRNA single-transgenic mice.</p

Phenotypic analysis of <i>Rhox3</i>-shRNA mice.

<p>(A) Testes weight of the control and <i>Rhox3</i>-shRNA mice at 6 weeks age derived from Lines 2, 11, 12 and 13. Values denote the mean ± S.E. (B) Sperm counts of the control and <i>Rhox3</i>-shRNA mice lines at 6 weeks of age. Values denote the mean ± S.E. (C) Representative image of testis from control and <i>Rhox3</i>-shRNA mice (Line 2) at 6 weeks age. (D) Representative images of hematoxylin and eosin (H&E) stained cross sections of testis and epididymis of control (Con) and <i>Rhox3</i>-shRNA (shRNA) mice. Scale bar: 100 μm. <i>Rhox3</i>-shRNA mice, <i>Rhox3</i>-shRNA;<i>Stra8</i>-iCre double-transgenic mice; Control mice, <i>Rhox3</i>-shRNA single-transgenic mice.</p

RHOX3 expression pattern in mice.

<p>Western blot analysis of adult tissue lysates (A), wild-type (WT) and <i>jsd</i> mutant mice testes lysates (B), and testes nuclear lysates from postnatal wild-type mice of the ages indicated (C). N, nuclear protein extract; C, cytoplasmic protein extract. β-ACTIN is the loading control. Histone-H3 is a chromatin marker to assess purity of the nuclear fraction.</p