Kisspeptin antagonists reveal kisspeptin 1 and kisspeptin 2 differential regulation of reproduction in the teleost, Morone saxatilis

The importance of kisspeptin in regulating vertebrate reproduction has been well established, but the exact mechanism continues to unfold. Unlike mammals, many lower vertebrates possess a dual kisspeptin system, Kiss1 and Kiss2. To decipher the roles of the kisspeptins in fish, we identified two potential kisspeptin antagonists, pep 234 and pep 359, by screening analogs for their ability to inactivate striped bass Kiss1 and Kiss2 receptors expressed in COS7 cells. Pep 234 (a mammalian KISS1 antagonist) antagonizes Kiss1r signaling activated by Kiss1 and Kiss2, and pep 359 (a novel analog) antagonizes Kiss2 activation of both receptors. In vitro studies using brain slices demonstrated that only Kiss2 can upregulate the expression of the hypophysiotropic gnrh1, which was subsequently diminished by pep 234 and pep 359. In primary pituitary cell cultures, the two antagonists revealed a complex network of putative endogenous and exogenous regulation by kisspeptin. While both kisspeptins stimulate Fsh expression and secretion, Kiss2 predominately induces Lh secretion. Pep 234 and 359 treatment of spawning males hindered sperm production. This effect was accompanied with decreased brain gnrh1 and gnrh2 mRNA levels and peptide content in the pituitary, and increased levels of pituitary Lh, probably due to attenuation of Lh release. Strikingly, the mRNA levels of arginine-vasotocin, the neurons of which in the preoptic area coexpress kiss2r, were dramatically reduced by the antagonists. Our results demonstrate differential actions of Kiss1 and Kiss2 systems along the hypothalamicpituitary axis and interactions with other neuropeptides, and further reinforce the importance of kisspeptin in the execution of spawning.The National Science Foundation program grant 1147118 and by research award IS-4499-12CR from the United States-Israel Binational Agricultural Research and Development Fund.http://www.biolreprod.org2016-09-30hb2016Mammal Research InstituteZoology and Entomolog

Dorsomorphin promotes survival and germline competence of zebrafish spermatogonial stem cells in culture.

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species

Inducible Sterilization of Zebrafish by Disruption of Primordial Germ Cell Migration.

During zebrafish development, a gradient of stromal-derived factor 1a (Sdf1a) provides the directional cue that guides the migration of the primordial germ cells (PGCs) to the gonadal tissue. Here we describe a method to produce large numbers of infertile fish by inducing ubiquitous expression of Sdf1a in zebrafish embryos resulting in disruption of the normal PGC migration pattern. A transgenic line of zebrafish, Tg(hsp70:sdf1a-nanos3, EGFP), was generated that expresses Sdf1a under the control of the heat-shock protein 70 (hsp70) promoter and nanos3 3?UTR. To better visualize the PGCs, the Tg(hsp70:sdf1a-nanos3, EGFP) fish were crossed with another transgenic line, Tg(kop:DsRed-nanos3), that expresses DsRed driven by the PGC-specific kop promoter. Heat treatment of the transgenic embryos caused an induction of Sdf1a expression throughout the embryo resulting in the disruption of their normal migration. Optimal embryo survival and disruption of PGC migration was achieved when transgenic embryos at the 4- to 8-cell stage were incubated at 34.5°C for 18 hours. Under these conditions, disruption of PGC migration was observed in 100% of the embryos. Sixty-four adult fish were developed from three separate batches of heat-treated embryos and all were found to be infertile males. When each male was paired with a wild-type female, only unfertilized eggs were produced and histological examination revealed that each of the adult male fish possessed severely under-developed gonads that lacked gametes. The results demonstrate that inducible Sdf1a expression is an efficient and reliable strategy to produce infertile fish. This approach makes it convenient to generate large numbers of infertile adult fish while also providing the capability to maintain a fertile brood stock

Results from three transplantation (T) experiments using 3-week-old spermatogonia cultured with dorsomorphin.

<p>Results from three transplantation (T) experiments using 3-week-old spermatogonia cultured with dorsomorphin.</p

Germline transmission of spermatogonia cultured for 6 weeks in the presence of dorsomorphin.

<p>(A) Photomicrograph showing the incorporation of DsRed-positive cultured spermatogonia (arrow) into the gonad of a recipient larva two weeks after transplantation. (B) Results of genomic PCR showing the presence of <i>piwil1-neo</i> sequences that were inherited by all of the F1 individuals (lanes 1 to 16) produced by a germline chimeric father. Negative control: genomic DNA template from a wild-type larva (lane 17); positive control: pPiwil1-neo plasmid DNA template (lane 18). (C) Dissection of a fertile adult male recipient fish showing that the transplanted DsRed-positive spermatogonia have proliferated and directed the formation of a pair of unequal sized testes (arrow) in the body. (C1) Inset shows the gonad under UV light revealing the presence of DsRed-positive cells. (D) Transverse section of testis from a fertile recipient fish showing active spermatogenesis. Sb: swim bladder; S: spermatozoa (arrow). Scale bar = 100 µm for A and 20 µm for D.</p

Dorsomorphin increased the growth of spermatogonia in 6 week cultures.

<p>(A) Addition of dorsomorphin significantly (p<0.001) increased the growth of spermatogonia maintained in culture for 6 weeks. (B) Merged bright field and UV photomicrographs showing a colony containing more than 100 DsRed-expressing spermatogonia. (C) RT-PCR analysis of RNA isolated from spermatogonia maintained for 6-weeks in culture in the presence of dorsomorphin, whole testis tissue and feeder cells alone showing expression of germ cell specific marker genes, <i>dazl</i>, <i>dnd</i>, <i>nanos3</i>, <i>vasa</i> and <i>piwil1</i> and spermatogonial markers <i>plzf</i> and <i>sox17.</i> RT: reverse transcription; Data points not sharing a letter (A, B, C, D) are significantly different by Bonferroni–Dunn tests. Scale bar = 20 µm.</p

Dorsomorphin inhibits the growth of FGSCs in 3 week cultures.

<p>(A) Addition of dorsomorphin significantly inhibits FGSC (A) colony formation and (B) cell proliferation in culture. Photomicrographs showing (C) a 3-week old FGSCs (arrows) cultured in the presence of 2 µM dorsomorphin and (D) a 3-week-old FGSC colony cultured in the absence of dorsomorphin. (E) RT-PCR analysis of RNA isolated from a 3-week FGSC culture grown in the presence or absence of dorsomorphin, whole ovaries and feeder cells alone. Dorsomorphin severely reduced the expression of germ cell specific marker genes, <i>dazl</i>, <i>dnd</i>, <i>nanos3</i>, <i>vasa</i> and <i>piwil1</i>. * indicates a significant difference by Student t-tests. Scale bar = 20 µm.</p

Feeder cells expressing zebrafish Lif, Fgf2 and Gdnf promote spermatogonial cell proliferation for 3 weeks in culture.

<p>Photomicrographs showing G418-selected spermatogonia that were initiated from <i>Tg(piwil1:neo);Tg(piwil1:DsRed)</i> zebrafish showing (A) 4-cell and (B) 15-cell colonies; DsRed expression in the (C) 4-cell and (D) 15-cell colonies. (E) OFC3LF and OFC3LG significantly enhanced spermatogonial cell proliferation in cultures maintained for 3 weeks while the mitogenic effect was lost after 6 weeks. * indicates a significant difference by Student t-tests. Scale bar = 20 µm.</p

Heat-treated transgenic embryos developed into infertile male adults.

<p>(A) No difference in appearance or overall size was observed between adult transgenic fish that developed from heat treated embryos and wild-type male. (B) No significant difference in body-weight of 3.5-month-old fish (n = 16 by random sampling) among heat-treat transgenic males, untreated transgenic males and wild-type males, and between the transgenic females and wild-type females. Data shared the same letter (A or B) are not significantly different from each other. Examination of gonadal tissue revealed that (C) A well-developed testis, C1, of untreated male transgenic fish. (D) A well-developed ovary, D1, of untreated female transgenic fish. (E) The gonads of heat-treated transgenic fish developed into a thin filament-like tissue, E1, surrounded by adipocytes. Photomicrograph showing (F) active spermatogenesis of the testis of untreated male transgenic fish, (G) a well-developed ovary with oocytes at different developmental stages of untreated female transgenic fish. (H) The gonad of heat-treated transgenic fish appears to be under-developed and surrounded with large amount of adipocytes without advanced gonadal structure or germ cells. WT: wild type; TG: transgenic; HT: heat treated. Scale bar: 1 cm for A, C–E and 50 µm for F–H. S: spermatozoa.</p

Dorsomorphin promotes the growth of spermatogonia in 3 week cultures.

<p>(A) RT-PCR results showing that feeder cells (OFC3LF+OFC3LG) express <i>bmp2a</i>, <i>bmp2b</i> and <i>bmp4</i>. (B) Addition of dorsomorphin significantly (p<0.001) enhanced spermatogonial cell proliferation in 3 week cultures. (C) Merged bright field and UV photomicrographs showing a colony containing approximately 65 DsRed-expressing spermatogonia. Data points not sharing a letter (A, B, C) are significantly different by Bonferroni–Dunn tests. Scale bar = 20 µm.</p