Expression and localization of the novel and highly conserved gametocyte-specific factor 1 during oogenesis and spermatogenesis

Abstract

Objective: To determine the onset of gametocyte-specific factor 1 (Gtsf1) expression in embryogenesis and its relation to Nobox; and to determine its localization during gonadal development and gametocyte maturation.

Design: Developmental animal study.

Setting: University reproductive biology laboratory.

Animal(s): Mice ranging in age from embryonic day 12.5 to 8 weeks.

Intervention(s): Polymerase chain reaction and quantitative polymerase chain reaction were performed to determine the onset of and relative messenger RNA expression. Western blot was performed to confirm protein expression and antibody specificity. In situ hybridization and immunohistochemistry were used determine localization of expression.

Main outcome measure(s): Gtsf1 messenger RNA expression levels during embryogenesis through adulthood in wild-type mice and in newborn Nobox knockout mice; GTSF1 expression and localization in postnatal mice.

Result(s): Gtsf1 functions downstream of Nobox and is highly expressed in embryonic male and female gonads, localizing to germ cells throughout development. GTSF1 expression is confined to the cytoplasm in all stages of postnatal oocyte maturation and to prespermatogonia during early postnatal testicular development.

Conclusion(s): The expression pattern of Gtsf1 and its high conservation suggests that it may play an important role in germ cell development. Further characterization of Gtsf1 may elucidate mechanisms involved in premature ovarian failure.

Figure 1

Gtsf1 expression in Wild-type and Nobox deficient female mice. Gtsf1 expression is downregulated 20-fold in Nobox deficient female mice compared to Wild-type female mice (p ≤ 0.011).

Figure 2

(A–C). Gtsf1 RNA and protein expression. Equal amounts of total RNA from 11 different adult tissues as indicated above (A) and from various embryonic time points (B) were reverse transcribed. Oligonucleotides complementary to Gtsf1 were used to amplify the corresponding RNA sequences. (C) Western blot with affinity-purified anti-GTSF1 antibodies were used to detect a 29 kDa band in Postnatal Day 3 ovaries and testis, but not liver.

Figure 3

GTSF1 expression in the ovaries (Panels A-D). Immunohistochemistry was performed on Newborn (Panel A & B) and 8 week old ovaries (Panel C & D). GTSF1 can be clearly detected (Panel B) in the cytoplasm of germ cell clusters (GCC) and primordial follicles (PF) in Newborn ovaries. At 8 weeks, GTSF1 can be clearly seen in all stages of developing oocytes, from primordial (PF) and primary follicles (PrF) (Panel D) to secondary (SF) and antral follicles (AF) (Panel C). GTSF1 expression in the testis (Panels E & F). Immunohistochemistry is performed on Postnatal day 10 (P10) testis. At Postnatal day 10, GTSF1 expression can be seen in primary spermatogonia (SG) but not in primary spermatocytes (SC). Scale bars, 50 μm