Results of artificial insemination of eggs derived from pure pejerrey mothers with sperm from three surrogate Patagonian pejerrey fathers (#1∼3; transplanted with pejerrey donor germ cells) and a control pure pejerrey father.

<p>Donor-derived germline transmission rates for each surrogate father were determined at 7 and 11 months after GCT; asterisks after the donor-derived germline transmission rate for 11 months indicate significant difference (Fisher's exact test) from the rate for the same father at 7 months. NA: not applicable</p

Changes in the gonado-somatic index of males (A) and females (B) subjected to heat (26°C) and Busulfan treatments (B0: Busulfan 0 mg/kg, controls; B30: 30 mg/kg, only females; B40: 40 mg/kg, only males) between 0 and 8 weeks.

<p>Columns with different letters vary significantly (ANOVA - Tukey test, <i>P</i><0.05).</p

Histological changes in the ovaries of females subjected to heat (26°C) and Busulfan treatment (30 mg/kg).

<p>Panels on the right are high magnifications of insets in the left panels. A,B) Start of the experiment (week 0; arrows show a prominent cyst of oogonia; note also oocytes at various stages indicating active oogenesis). C,D) 4 weeks (note the absence of prominent cysts of oogonia, degenerating perinucleolar oocytes (arrowhead), and macrophage phagocytic activity indicated by deposition of yellowish-brown pigments (arrow). E,F) 8 weeks (note the absence of oogonia and other types of GCs). G,H) 24 weeks (note the absence of GCs, disorganized ovigerous lamellae (arrows), and hypertrophy of the tunica albuginea). Scale bars indicate 100 µm (A, C, E, and G) and 20 µm (B, D, F and H).</p

Surrogate Production of Eggs and Sperm by Intrapapillary Transplantation of Germ Cells in Cytoablated Adult Fish

<div><p>Germ cell transplantation (GCT) is a promising assisted reproductive technology for the conservation and propagation of endangered and valuable genetic resources. In teleost fish, GCT in adult gonads has been achieved only in male recipients, limiting greatly the usefulness of this technique in situations where both sexes need equal and timely attention for conservation and/or propagation. Here we describe a simplified GCT approach that ultimately leads to production of donor-derived eggs and sperm in considerably short time. Donor germ cells isolated from young pejerrey <i>Odontesthes bonariensis</i> (Atherinopsidae) were transplanted non-surgically through the genital papilla into the sexually mature gonads of Patagonian pejerrey <i>O. hatcheri</i> recipients whose gonads have been depleted of endogenous GCs by heat (26°C) and chemical treatment (four doses of Busulfan at 30 mg/kg and 40 mg/kg for females and males, respectively). Transplanted spermatogonial and oogonial cells were able to recolonize the recipients' gonads and produce functional donor origin eggs and sperm within 7 months from the GCT. We confirmed the presence of donor-derived gametes by PCR in 17% and 5% of the surrogate <i>O. hatcheri</i> fathers and mothers, respectively. The crosses between surrogate fathers and <i>O. bonariensis</i> mothers yielded 12.6–39.7% pure <i>O. bonariensis</i> and that between a surrogate mother and an <i>O. bonariensis</i> father yielded 52.2% pure <i>O. bonariensis</i> offspring. Our findings confirm that transplantation of germ cells into sexually competent adult fish by non-surgical methods allows the production of functional donor-derived eggs and sperm in a considerably short time. The methods described here could play a vital role in conservation and rapid propagation of endangered fish genetic resources.</p></div

PCR analysis of eggs from 20 female recipients 7 months after germ cell transplantation.

<p>The primers used in the analysis were based on an <i>O. bonariensis</i>-specific sequence (A), an <i>O. hatcheri</i>-specific sequence (B), and β-actin (C) as a template control. Control lanes include pure <i>O. bonariensis</i> (C1) and an <i>O. hatcheri</i> (C2) eggs. Donor-derived <i>O. bonariensis</i> eggs were detected in one surrogate <i>O. hatcheri</i> recipient shown in lane 8.</p

Results of artificial insemination of eggs derived from a surrogate Patagonian pejerrey mother (#1; transplanted with pejerrey donor germ cells) and from a control pure pejerrey mother with sperm from a pure pejerrey father.

<p>Donor-derived germline transmission rates determined for the same surrogate mother at 7 and 11 months after GCT were not significantly different (Fisher's exact test). NA: not applicable.</p

PCR analysis of sperm from 17 male recipients 7 months after germ cell transplantation.

<p>The primers used in the analysis were based on an <i>O. bonariensis</i>-specific sequence (A), an <i>O. hatcheri</i>-specific sequence (B), and β-actin (C) as a template control. Control lanes include pure <i>O. bonariensis</i> (C1) and <i>O. hatcheri</i> (C2) sperm. Donor-derived <i>O. bonariensis</i> spermatozoa were detected in the sperm of three surrogate <i>O. hatcheri</i> recipients shown in lanes 4, 9, and 17.</p

Fate of PKH 26-labeled donor germ cells in recipient ovaries between 2 and 24 weeks after germ cell transplantation.

<p>A,B) Cryostat section of a transplanted ovary at 2 weeks showing donor germ cells randomly distributed throughout the ovarian lamellae (circles; B is a fluorescent view of bright field A). C,D) Cryostat sections of a transplanted ovary at 4 weeks showing the donor germ cells (presumably oogonia) forming aggregations (highlighted; D is a fluorescent view of bright field C). E,F) Whole-mount preparation of oocytes from a transplanted female at 6 months showing the presence of fully differentiated donor-derived oocytes (characterized by retention of fluorescent label). G,H) Whole-mount preparation of oocytes from a non-transplanted control female. Scale bars indicate 20 µm (A, B, C and D) and 100 µm (E, F, G and H).</p

Histological criteria for classification of gonadal integrity/degeneration in adult Patagonian pejerrey <i>Odontesthes hatcheri</i> (modified from Majhi <i>et al</i>. 2009).

<p>Histological criteria for classification of gonadal integrity/degeneration in adult Patagonian pejerrey <i>Odontesthes hatcheri</i> (modified from Majhi <i>et al</i>. 2009).</p

Fate of PKH-26-labeled donor germ cells in recipient testes between 4 and 24 weeks after germ cell transplantation.

<p>A,B) Cryostat section of a non-transplanted, control testis at 4 weeks showing the approximate location of the blind end of the spermatogenic lobules (B is a high magnification of the box in A). C,D) Cryostat section of a transplanted testis at 4 weeks showing the presence of transplanted GCs at the blind end of the spermatogenic lobules (arrow; D is a high magnification of the box in C). E) Whole-mount preparation of a non-transplanted control testis at 8 weeks. F) Whole-mount preparation of a transplanted testis at 8 weeks showing the presence of donor-derived GCs (arrows) along the length of the gonad. G-I) Cryostat (G), corresponding HE (H) and merged (I) sections of a transplanted testis at 6 months showing differentiation of the donor-derived cells along the spermatogenic lobules towards the efferent ducts. Scale bars indicate 100 µm (A, C, E, F, G, H and I) and 20 µm (B and D).</p