Dynamics of INSL3 Peptide Expression in the Rodent Testis1

Abstract

The Leydig cell-specific factor insulin-like peptide 3 (INSL3) is involved in testicular descent during embryo development, and has been suggested to regulate spermatogenesis and bone metabolism in the adult. Using a new, sensitive assay specific for rodent INSL3, we have mapped the secretion of INSL3 into peripheral blood in mice and during postnatal male rat development (in female rats, circulating INSL3 is at the level of detection). Maximum INSL3 is measured at Postnatal Day (PD) 40 in the rat and decreases to a significantly lower, stable value by PD60, indicating an "overshoot" effect in the establishment of Leydig cell functionality during the first wave of spermatogenesis. Aging rats ( approximately 24 mo) have markedly reduced circulating INSL3 levels, as do humans. Treatment of young adult rats with ethane dimethylsulfonate (EDS) leads to loss of mature Leydig cells and no detectable INSL3 in peripheral blood. INSL3 can be detected first at Day 27 after EDS treatment, returning to near normal levels by Day 37. Both primary rat Leydig cells and the mouse MA-10 tumor cell line secrete substantial amounts of INSL3 into the culture media in a constitutive manner, unregulated by common effectors, including hCG. Analysis of different testicular fluid compartments shows highest INSL3 concentration in the interstitial fluid (391.4 +/- 47.8 ng/ml). However, INSL3 evidently traverses the blood-testis barrier to enter the seminiferous compartment, rete testis, and epididymis in sufficient concentration to be able to address the specific INSL3 receptors (RXFP2) on post-meiotic germ cells and in the epididymis.

Brief maternal exposure of rats to the xenobiotics dibutyl phthalate or diethylstilbestrol alters adult-type Leydig cell development in male offspring

“…21 As Leydig cells differentiate during puberty, concomitant with the large and initially not well regulated pulses of LH from the pituitary, they initially give rise to an overproduction of INSL3 as a measure of Leydig cell differentiation status. Unlike testosterone, whose enzymatic production is acutely regulated by feedback from the pituitary, INSL3 is constitutively expressed.…”

“…20 INSL3 in blood was measured using a rodent-specific time-resolved fluorescent immunoassay, exactly as previously described. 21 Luteinising hormone (LH) was estimated using a commercial rat LH ELISA kit (USCN Life, Wuhan, China), according to the manufacturer's protocol. The range of detection for this assay was 0.78 mIU ml 21 -50 mIU ml 21 .…”

“…21 Luteinising hormone (LH) was estimated using a commercial rat LH ELISA kit (USCN Life, Wuhan, China), according to the manufacturer's protocol. The range of detection for this assay was 0.78 mIU ml 21 -50 mIU ml 21 . The assay was specific for natural or recombinant rat LH and had no reported cross-reactivity with other related proteins.…”

Relaxin family peptide systems and the central nervous system

“…Recently several studies have described immunoassays capable of measuring circulating INSL3 levels [53,54]. In human males, the circulating levels of INSL3 are relatively high; about 1% of testosterone levels, but much greater than that of the sertoli cell hormone, inhibin-B [54].…”

“…In conscious rats, MS infusions of R3/I5 and the RXFP3 antagonist R3(BD23-27)R/I5 [100], differentially modulated hippocampal theta activity. In a behavioral paradigm that investigates theta rhythm-dependent spatial working memory, infusions of R3(BD23-27)R/I5 impaired rats performance of the task in a dose-dependent manner [53] (Fig. 7).…”