Rhox13 is required for a quantitatively normal first wave of spermatogenesis in mice

Abstract

We previously described a novel germ cell-specific X-linked reproductive homeobox gene (Rhox13) that is upregulated at the level of translation in response to retinoic acid (RA) in differentiating spermatogonia and preleptotene spermatocytes. We hypothesize that RHOX13 plays an essential role in male germ cell differentiation, and have tested this by creating a Rhox13 gene knockout (KO) mouse. Rhox13 KO mice are born in expected Mendelian ratios, and adults have slightly reduced testis weights, yet a full complement of spermatogenic cell types. Young KO mice (at ~7-8 weeks of age) have a ≈50% reduction in epididymal sperm counts, but numbers increased to WT levels as the mice reach ~17 weeks of age. Histological analysis of testes from juvenile KO mice reveals a number of defects during the first wave of spermatogenesis. These include increased apoptosis, delayed appearance of round spermatids and disruption of the precise stage-specific association of germ cells within the seminiferous tubules. Breeding studies reveal that both young and aged KO males produce normal-sized litters. Taken together, our results indicate that RHOX13 is not essential for mouse fertility in a controlled laboratory setting, but that it is required for optimal development of differentiating germ cells and progression of the first wave of spermatogenesis.

Figure 1. Generation of Rhox13 KO mice

(A) The Rhox13 gene is comprised of three exons (shown in boxes), as is typical of most homeobox genes. We created a KO allele by deleting exon 2, and a single loxP site remains (red triangle). (B) PCR amplification using primers on either side of exon 2 distinguishes KO from WT alleles. MW = molecular weight ladder, and NTC = no template control. (C–D) IHC was done to verify loss of RHOX13 protein (brown staining) in P>60 adult testes. Shown are stage VII-VIII seminiferous tubule sections from WT (C) and KO (D) mice. Sections were counterstained with hematoxylin, and inset (in D) is a no primary antibody control. Spg = spermatogonia, Pl = preleptotene spermatocytes, P = pachytene spermatocytes, RS = round spermatids, Ser = Sertoli cells. (E–F) IIF was done to localize RHOX13 (in green) in WT (E) and KO (F) P56 testes. DNA is stained with DAPI (blue), and F-actin is stained with fluorescently-conjugated phalloidin (red). Scale bar = 50 µm.

Figure 2. Histological abnormalities of adult (P>60) testes from Rhox13 KO on a mixed genetic background

(A–D) Bouin’s-fixed KO sections are stained with H&E and abnormal tubules are indicated by asterisks (★ = only containing condensed spermatids and Sertoli cells; ★★ = lacking spermatids; ★★★ = lacking spermatids and almost all spermatogonia). Scale bar = 50 µm.

Figure 3. Fecundity results from long-term breeding trial of Rhox13 WT and KO mice on mixed genetic background

(A) The average litter size and days to first litter were not statistically different between Rhox13 WT and KO males. (B) In a graphical representation of time between litters, a horizontal gray line represents the average. Statistical significance is indicated by an asterisk (P=0.008).

Figure 4. Developmental delays and histological abnormalities during the first wave of spermatogenesis in backcrossed Rhox13 KO mice

There were no significant differences in the numbers of preleptotene spermatocytes at P8 (A–B) or leptotene (Lep) and zygotene (Zyg) spermatocytes at P12 (C–D). In contrast, P20 KO testes had significantly reduced numbers of round spermatids (RS) (E–F). Asterisk indicates significance at P=0.0002. Scale bar = 50 µm.

Figure 5. Germ cell proliferation decreases in backcrossed KO testes during the first wave of spermatogenesis

(A–D) Immunostaining was done to detect BrdU (green) and DDX4 (red), and nuclei were labeled with DAPI (blue) in testes from P8 (A–B) and P20 (C–D) WT (A, C) and KO (B, D) mice. Ages are indicated on each panel. Scale bars = 30 µm.

Figure 6. Increased germ cell apoptosis in backcrossed KO testes during the first wave of spermatogenesis

(A–F) Immunostaining was done to detect c-PARP1 (green), and phalloidin labeled F-actin (red). The c-PARP1+ cells were counted at each age and in each genotype, and the results are presented for each age below their respective panels as c-PARP1+ cells per mm² of seminiferous cord (P8, P12) or tubule (P20). Asterisk indicates statistical significance (P<0.05). Scale bar = 30 µm.