Neuropeptide FF indirectly affects testicular morphogenesis and functions in medaka

Testicular morphogenesis and functions are considered to be under the control of neural and endocrine systems. However, the available literature is mainly limited to mammals, and it remains unclear how they are regulated in teleost species. Here, we demonstrated that neuropeptide FF (NPFF) in the brain is responsible for the follicle-stimulating hormone expression in the pituitary, which facilitates the testicular morphogenesis and androgen synthesis, and subsequently contributes to successful spermatogenesis. The present findings give us important insights into the neuroendocrine regulatory mechanisms underlying the testicular morphogenesis and functions in teleosts.

Testicular morphogenesis and functions are considered to be under the control of neural and endocrine systems. However, the available literature is mainly limited to mammals, and it remains unclear how they are regulated in teleost species. Here, we demonstrated that neuropeptide FF (NPFF) in the brain is responsible for the follicle-stimulating hormone expression in the pituitary, which facilitates the testicular morphogenesis and androgen synthesis, and subsequently contributes to successful spermatogenesis. The present findings give us important insights into the neuroendocrine regulatory mechanisms underlying the testicular morphogenesis and functions in teleosts.
testis j neuropeptide FF j teleost Testicular morphogenesis and functions are generally considered to be regulated by the hypothalamic-pituitary-gonadal axis in vertebrates (1)(2)(3). In mammals, it is widely accepted that gonadotropin-releasing hormone (GnRH) from GnRH neurons in the hypothalamus induces the release of follicle-stimulating hormone (FSH) from the pituitary, which facilitates spermatogenesis in testis (4). However, in teleosts, hypophysiotropic GnRHknockout (KO) males show normal testes in medaka (5). It has also been reported that FSH-or FSH receptor-KO males also show normal testes in teleosts (5)(6)(7). Thus, the regulatory mechanism of testicular functions in teleosts has not been well understood. Here, we focused on an RFamide peptide as a candidate regulator of testicular functions, since it has been suggested that besides GnRH, some RFamide neuropeptides (neuropeptides with C-terminal Arg-Phe-NH 2 motif) are involved in spermatogenesis or Sertoli cell maturation in mammals (8,9). Furthermore, this neuropeptide family is conserved among vertebrate species (10). We hypothesized that RFamide neuropeptides play a key role in testicular functions in teleosts and analyzed the functions of an RFamide called neuropeptide FF (NPFF) in a teleost medaka by using npff-KO males.

Results
We found that npff-KO (npff À/À ) males showed a low fertilization rate (Fig. 1A) and small testes at 5 to 6 months post fertilization (mpf) (Fig. 1 B, Upper Row, and C). We observed the testicular architecture in hematoxylin and eosin (HE)-stained preparations. We found that npff À/À testes showed normal testicular structure (Fig. 1B, Center Row), although they were smaller than those of wild type (WT), particularly at 5 to 6 mpf ( Fig. 1 B, Upper Row, and C). In addition, in situ hybridization (ISH) revealed that odf3, a mature sperm marker (11), was expressed even in the npff À/À testes at all ages (Fig. 1B, Bottom Row), indicating that npff À/À testes can produce mature sperms regardless of their small size. We then performed quantitative PCR (qPCR) analysis and found that the expression level of odf3 in npff À/À testes was lower than that in WT (Fig. 1D). To further examine the effects of npff loss on the testicular functions, we analyzed the expression of Sertoli/Leydig cell-specific genes ( Fig. 1 E-J). npff À/À showed a significant increase in 3bhsd and cyp17 expressions ( Fig. 1 H and I), which are genes for androgen synthetic enzymes. We also examined the blood concentration of 11-ketotestosterone (11-KT), which is an essential sex steroid hormone for spermatogenesis in fish (12). Since WT male medaka showed time-of-day changes in 11-KT concentrations, higher in the evening than in the morning (Fig. 1K), we collected blood in the evening and compared 11-KT concentrations between WT and npff À/À . The 11-KT concentration of 5 to 6 mpf npff À/À blood was lower than that of WT (Fig. 1L).

Discussion
The present study demonstrates that NPFF plays a crucial role in testicular morphogenesis and functions by increasing the expression of fshb in the pituitary, because npff À/À males showed a low fertilization rate (Fig. 1A), small testes ( Fig. 1  B and C), and low fshb expression in the pituitary (Fig. 2C). We have previously shown that NPFF is specifically expressed in the terminal nerve in the teleost brain and the NPFF neurons project their axons broadly in the brain but not the pituitary (13,14), and here we showed that NPFF receptors (gpr147/74-2) expressing neurons in the POm projected to the pituitary (Fig. 2F). Taken together, it is conceivable that NPFF modulates fshb expression via neurons expressing NPFF receptors in the POm (Fig. 2G). It should be noted that FSH-or FSH receptor-KO males have been reported to demonstrate normal testes (3)(4)(5). In the present study, experiments using older fish (at 5 to 6 mpf) than those in the previous studies (at ∼3 mpf) enabled us to find that FSH is involved in the testicular functions, at least in medaka.
Interestingly, 11-KT levels of WT males showed the difference between morning and evening (Fig. 1K), suggesting that there is a diurnal fluctuation of 11-KT levels in WT males. Also, our results showed that npff À/À males showed lower 11-KT levels in the evening (Fig. 1K), lower expression of odf3 (Fig. 1D), and a lower fertilization rate (Fig. 1A), which suggests that sufficient levels and/or diurnal fluctuation of testosterone play a role in appropriate spermatogenesis in teleost species. It should be noted that npff À/À showed higher expression of 3bhsd/cyp17 (Fig. 1 H and I), which contradicts the results of lower 11-KT levels. It is conceivable that these steroidogenic genes are up-regulated to compensate for the deficiency of FSH signaling caused by npff KO, but it is insufficient to reinstate 11-KT level due to the small size of the KO's testis.
In summary, our present study suggests that NPFF affects testicular morphogenesis and functions, as illustrated in Fig.  2G. Since we demonstrated NPFF function in males, further analysis of NPFF in females will also help understanding teleost reproduction. In teleosts, the neuroendocrine mechanism  regulating testicular function has been relatively unraveled compared to that of the ovary. Our study may advance the understanding of brain functions for successful reproduction in teleost males.

Materials and Methods
We kept and used d-rR WT, npff À/À and fshb À/À medaka (Oryzias latipes) as described by Umatani et al. (14) and Takahashi et al. (5). All of the experiments were conducted in accordance with the protocols approved by the animal care and use committee of the Graduate School of Science, University of Tokyo (permission no. 20-6). Detailed procedures for other analyses are described in SI Appendix.
Data, Materials, and Software Availability. All of the study data are included in the article and/or supporting information.