Targeted Gene Disruption in Zebrafish Reveals Noncanonical Functions of LH Signaling in Reproduction

Abstract

The pivotal role of gonadotropin signaling in regulating gonadal development and functions has attracted much research attention in the past 2 decades. However, the precise physiological role of gonadotropin signaling is still largely unknown in fish. In this study, we have established both LH β-subunit (lhb) and LH receptor (lhr) knockout zebrafish lines by transcription activator-like effector nucleases. Intriguingly, both homozygous lhb and lhr mutant male fish are fertile. The fertilization rate, sperm motility, and histological structure of the testis were not affected in either lhb or lhr mutant males. On the contrary, homozygous lhb mutant females are infertile, whereas homozygous lhr mutant females are fertile. Folliculogenesis was not affected in either lhb or lhr mutants, but oocyte maturation and ovulation were disrupted in lhb mutant, whereas only ovulation was affected in lhr mutant. Differential expression of genes in the ovary involved in steroidogenesis, oocyte maturation, and ovulation was found between the lhb and lhr mutants. These data demonstrate the essential role of LH signaling in oocyte maturation and ovulation, and support the notion that LH acts through the FSH receptor in the absence of LH receptor. Moreover, the defects of lhb mutant could be partially restored by administration of human chorionic gonadotropin. This in vivo evidence in the present study demonstrates, for the first time in any vertebrate species, that LH signaling is indispensable in female reproduction but not in male reproduction. LH signaling is demonstrated to control oocyte maturation and ovulation in the ovary.

Disruption of Zebrafish Follicle-Stimulating Hormone Receptor (fshr) But Not Luteinizing Hormone Receptor (lhcgr) Gene by TALEN Leads to Failed Follicle Activation in Females Followed by Sexual Reversal to Males

“…This agrees with a similar study published recently (67). Our further experiments on early stages of development revealed no difference either between lhcgr mutant and the control in terms of follicle activation or puberty onset.…”

Fsh Stimulates Spermatogonial Proliferation and Differentiation in Zebrafish via Igf3

“…Fsh is the main gonadotropin driving the initial stages of spermatogenesis in fish (33, 34, 44 -48), by regulating both Leydig and Sertoli cell activities until spawning. Further support for this conclusion is provided by the observation that loss-of-function of the lhcgr (49) or lhb (49 -50) gene in zebrafish results in female but not in male infertility (49 -50), suggesting that Lh bioactivity is not required for spermatogenesis in zebrafish while loss-offunction of the fshb gene in male zebrafish delayed puberty (50). However, male infertility was observed when both fshr and lhcgr genes were knocked out (51).…”

A short evolutionary history of FSH-stimulated spermatogenesis

“…57 Another divergence from the mammalian system is that Fshr and Lhr are expressed in fishes both in Leydig and Sertoli cells. 58,59 Recent studies on gonadotrophin and gonadotrophin receptor knockouts in zebrafish using the TALE technique [60][61][62] have elucidated the marked special features of gonadotrophin action that diverge from mammals. Neither lhb nor lhr knockouts affected testicular histology, fertilization rate or sperm motility in male fishes, indicating that Lh signaling, unlike in mammals, is not important for the fertility of the male zebrafish.…”