Genetic disruption of the baculum compromises the ability of male mice to copulate

Abstract

The baculum, a bone in the penis of many mammal species, shows an astonishing level of morphological divergence between species. Despite hundreds of years of interest, biologists have been unable to directly test its function. The goal of the current study is to uncover molecular details that could allow selective disruption of the baculum while allowing normal sexual differentiation and skeletal development. We compare patterns of androgen receptor binding and single cell gene expression in the developing penis, forelimbs and hindlimbs of mice. We identified chondrocytes in all three tissue types, but those from the developing penis show several unique features, including a population of chondrocytes that express both Runt-related transcription factor 2 (Runx2) and Androgen receptor (Ar). By combining a Runx2-Cre allele with a floxed Ar allele in mice, we selectively knocked out androgen signaling in late chondrocytes, resulting in a range of defects in baculum morphology. Males with the most disrupted bacula were unable to copulate, and their bacula appears to be disconnected from the corpus cavernosum muscle. Our study provides insights into the diversity of molecular mechanisms leading to bone and offers the first opportunity to directly test the function of the baculum.

Fig 1. Integrated Data of scRNA-seq data from three tissue types.

Top row: general classication of cell clusters across all three samples, determined by inspection of marker genes. Rows 2-5: Expression of Transcription factor SOX-9 (Sox9), collagen type II alpha 1 (Col2a1), Runx2, and Ar. The words “early” and “late” indicate genes that are turned on early/late in chondrogenesis. Expression levels go from blue (relatively low expression) to yellow (relative high expression). Gray points are "ghost points", indicating cells that were present in one of the other tissues sampled and only included to provide spatial context. Dashed rectangle surrounds all chondrocytes sampled from Penis, and is the area highlighted in Fig 2.

Fig 2. Close-up view of dashed rectangle in Fig 1, including only the area that surrounds all chondrocytes detected from Penis.

Rows the same as in Fig 1. The two red circles indicate the two separate clusters of chondrocytes in the penis sample, and corresponding areas in the forelimb and hindlimb. In the penis sample, the upper right circle indicates chondrocyte cells that overlap with forelimb and hindlimb chondrocytes. These chondrocytes do not express Ar. The lower left circle indicates chondrocytes that cluster separately from forelimb and hindlimb chondrocytes and do express Ar.

Fig 3. Sagittal sections of a wild type (top row) and baculum-reduced (bottom row) male.

Bone is stained black with Von Kossa (left column), while cartilage is stained red with Saf-O (right column). Penises are oriented with distal end to the right. The proximal end of the main bone forms via endochondral ossification (E), while the distal shaft forms via intramembranous ossification (I). The bony baculum is capped by a cartilaginous mass (C) sometimes referred to as the MUMP. The corpus cavernosum muscle (M) engulfs and attaches to the proximal end of the bony baculum. Red boxes and arrows highlight penile spines that surround the penis.

Fig 4. Multi-paneled figure.

The panels show microCT scanned images of male animals in our study. The scale ranging from a dark purple to a bright yellow indicates severity of the Androgen Receptor Knock-out (ARKO) phenotype. Dark purple indicates wildtype individuals, turquoise indicates relatively mild ARKO phenotype, and bright yellow indicates the severe ARKO phenotype. Two microCT scans are represented for each individual. The top scan is a dorsal view. The bottom scan is a lateral view. Numbers indiciate specimen ID's.

Fig 5. Linear discriminant analysis showing the femora of baculum-reduced males ranges from female-like to male-like.

LD1 = linear discriminant dimension 1, calculated by analyzing only wildtype (WT) males and females, and then projecting ARKO (KO) males into that space. Distal ends of femora towards right of figure, proximal towards left.