Male contraception via simultaneous knockout of α1A-adrenoceptors and P2X1-purinoceptors in mice

Abstract

Therapeutic targets for male contraception are associated with numerous problems due to their focus on disrupting spermatogenesis or hormonal mechanisms to produce dysfunctional sperm. Here we describe the dual genetic deletion of α1A-adrenergic G protein-coupled receptors (adrenoceptors) and P2X1-purinoceptor ligand gated ion channels in male mice, thereby blocking sympathetically mediated sperm transport through the vas deferens during the emission phase of ejaculation. This modification produced 100% infertility without effects on sexual behavior or function. Sperm taken from the cauda epididymides of double knockout mice were microscopically normal and motile. Furthermore, double knockout sperm were capable of producing normal offspring following intracytoplasmic sperm injection into wild-type ova and implantation of the fertilized eggs into foster mothers. Blood pressure and baroreflex function was reduced in double knockout mice, but no more than single knockout of α1A-adrenoceptors alone. These results suggest that this autonomic method of male contraception appears free of major physiological and behavioral side effects. In addition, they provide conclusive proof of concept that pharmacological antagonism of the P2X1-purinoceptor and α1A-adrenoceptor provides a safe and effective therapeutic target for a nonhormonal, readily reversible male contraceptive.

Keywords: ATP; noradrenaline; sympathetic neurotransmission.

Fig. 1.

Breeding and gross morphological effects of α_1A (^−/−)/P2X1 (^−/−) double knockout in male mice. (A) Breeding strategy. (B) Genotyping by PCR. (C) Photos of vas deferens from wild-type and double knockout α_1A (^−/−)/P2X1 (^−/−) mice. (D) Photos of whole prostates from wild-type and double knockout α_1A (^−/−)/P2X1 (^−/−) mice.

Fig. 2.

Vas deferens histochemistry and sperm viability. P2X1-purinoceptor immunohistochemistry of cross-sections of vas deferens from (A) wild-type male mice (n = 3) and (B) double knockout α_1A (^−/−)/P2X1 (^−/−) male mice (n = 3). X-gal staining for β-galactosidase in cross-sections of vas deferens from (C) wild-type male mice (n = 3) and (D) double knockout α_1A (^−/−)/P2X1 (^−/−) male mice (n = 3). SPG histochemical fluorescence of cross-sections of vas deferens from (E) wild-type male mice (n = 3) and (F) double knockout α_1A (^−/−)/P2X1 (^−/−) male mice (n = 3). (Scale bars, 100 μm.) (G) Fertilization of wild-type ova in vitro by intracytoplasmic injection of sperm extracted from cauda epididymides of male double knockout α_1A (^−/−)/P2X1 (^−/−) mice; the photomicrograph was taken at 3 d postinjection. (H) Weanlings (black/agouti) 7 wk after implantation of fertilized ova into a foster mother (white).

Fig. 3.

Vas deferens contractility and electrical activity. (A) Typical recording of contractile responses to electrical field stimulation of isolated vasa deferentia taken from wild-type, α_1A (^−/−) single knockout, P2X1 (^−/−) single knockout, and double knockout α_1A (^−/−)/P2X1 (^−/−) mice. (B) Mean frequency–response curve to electrical field stimulation of isolated vasa deferentia taken from wild-type (n = 7), α_1A (^−/−) single knockout (n = 10), P2X1 (^−/−) single knockout (n = 9), and double knockout α_1A (^−/−)/P2X1 (^−/−) (n = 21) mice. (C) Mean frequency–response curve to electrical field stimulation of isolated vasa deferentia taken from double knockout α_1A (^−/−)/P2X1 (^−/−) mice in the absence and presence of BMY 7378 (n = 7). (D) Mean contractile response to 80 mM KCl of isolated vasa deferentia taken from wild-type (n = 10; open columns) and double knockout α_1A (^−/−)/P2X1 (^−/−) (n = 21; filled columns) mice. (E) Mean concentration–response curve to exogenous administration of noradrenaline (NA) in isolated vasa deferentia taken from wild-type (n = 6), α_1A (^−/−) single knockout (n = 10), P2X1 (^−/−) single knockout (n = 10), and double knockout α_1A (^−/−)/P2X1 (^−/−) (n = 5) mice. (F) Mean concentration–response curve to exogenous administration of noradrenaline in isolated vasa deferentia taken from double knockout α_1A (^−/−)/P2X1 (^−/−) mice in the absence and presence of BMY 7378 (n = 6). (G) Mean concentration–response curve to exogenous administration of ATP in isolated vasa deferentia taken from wild-type (n = 10), α_1A (^−/−) single knockout (n = 7), P2X1 (^−/−) single knockout (n = 4), and double knockout α_1A (^−/−)/P2X1 (^−/−) (n = 5) mice. (B–G) Y-axes: Force (g.s) = integral of force (g) × time (s). Error bars represent SEM. (H) Representative intracellular spontaneous electrical activity recordings of vas deferens smooth muscle from wild-type and double knockout α_1A (^−/−)/P2X1 (^−/−) mice. (I) Representative intracellular electrical responses to exogenous administration of ATP (upward arrow, 1 mM) of vas deferens smooth muscle from wild-type and double knockout α_1A (^−/−)/P2X1 (^−/−) mice. (J) Representative intracellular electrical responses to exogenous administration of K⁺ (upward arrow, 20 mM) of vas deferens smooth muscle from wild-type and double knockout α_1A (^−/−)/P2X1 (^−/−) mice. (I, J) Downward arrows signify washout.

Fig. 4.

Cardiovascular responses in double knockout α_1A (^−/−)/P2X1 (^−/−) mice. (A) Resting systolic arterial blood pressure (BP) and (B) resting pulse rate as measured by the tail cuff method in conscious wild-type (n = 8), α_1A (^−/−) single knockout (n = 11), P2X1 (^−/−) single knockout (n = 11), and double knockout α_1A (^−/−)/P2X1 (^−/−) (n = 14) male mice. Asterisks represent a significant difference, *P , 0.05, ***P < 0.001. Mean effect of A61603 (0.2–2.0 μg/kg) in wild-type (n = 6; open circles) and double knockout α_1A (^−/−)/P2X1 (^−/−) (n = 5; filled squares) male mice on (C) carotid artery pressure and (D) heart rate. bpm, beats per min; MAP, mean arterial pressure. Mean effect of phenylephrine (5–100 μg/kg) in wild-type (n = 6; open circles) and double knockout α_1A (^−/−)/P2X1 (^−/−) (n = 5; filled squares) male mice on (E) carotid artery pressure and (F) heart rate. Mean changes to isoprenaline (3 μg/kg) and atropine (1 mg/kg) in wild-type (n = 6) and double knockout α_1A (^−/−)/P2X1 (^−/−) (n = 5) male mice to (G) mean arterial pressure and (H) heart rate. Error bars represent SEM.