Evidence for Almost Complete Sex-reversal in Bovine Freemartin Gonads: Formation of Seminiferous Tubule-like Structures and Transdifferentiation into Typical Testicular Cell Types

Abstract

Abstract. During mammalian sex determination of XY fetuses, SRY induces SOX9 in Sertoli cells, resulting in formation of testes with seminiferous tubules, interstitial Leydig cells and peritubular myoid cells. Meanwhile XX fetuses without SRY develop ovaries. In cattle, most XX heifers born with a male twin, so-called freemartins, develop nonfunctioning ovaries and genitalia with an intersex phenotype. Interestingly, freemartins sometimes develop highly masculinized gonads with seminiferous tubule-like structures despite the absence of SRY. However, in these cases, the degree of masculinization in each gonadal somatic cell type is unclear. Here, we report a rare case of a freemartin Japanese black calf with almost complete XX sexreversal. Gross anatomical analysis of this calf revealed the presence of a pair of small testis-like gonads with rudimentary epididymides, in addition to highly masculinized genitalia including a pampiniform plexus, scrotum and vesicular gland. Histological and immunohistochemical analyses of these masculinized gonads revealed well-defined seminiferous tubulelike structures throughout the whole gonadal parenchyma. In epithelia of these tubules, SOX9-positive supporting cells (i.e., Sertoli cells) were found to be arranged regularly along the bases of tubules, and they were also positive for GDNF, one of the major factors for spermatogenesis. 3β-HSD-positive cells (i.e., Leydig cells) and SMA-positive peritubular myoid cells were also identified around tubules. Therefore, for the first time, we found the transdifferentiation of ovarian somatic cells into all testicular somatic cell types in the XX freemartin gonads. These data strongly support the idea of a high sexual plasticity in the ovarian somatic cells of mammalian gonads.

Immunolocalization of Cytoskeletal Proteins in the Testes of Two Asian Cervids: Water Deer (<i>Hydropotes inermis</i>) and Reeves’ Muntjac (<i>Muntiacus reevesi</i>)

“…The sections were then irradiated for 20 min with each primary antibody in the microwave followed by the instruction manual of the microwave processor. The primary antibodies used in this study were the following: monoclonal anti-porcine stomach desmin in mouse (1:50, code M724, DAKO), polyclonal anti-calf lens vimentin in rabbit (1:100, code VIP, MEDAC, Hamburg, Germany) and monoclonal anti-human α-smooth muscle actin (α-SMA) in mouse (1:1,000, Clone 1A4, Sigma Chemical Co., St. Louis, MO, U.S.A.) [17,20]. For secondary antibodies, the sections with biotinylated anti rabbit IgG in goat (1:200, BA-1000, Vector Laboratories Inc.) or biotinylated anti mouse IgG in goat (1:200, BA-9200, Vector Laboratories Inc.) were irradiated for 5 min, and then the sections were incubated with ABC-kit (1:2, PK-6100, Vectastain Elite ABC kit, Vector laboratories Inc.) for 5 min.…”

A Rapid Multiplex PCR Method for the Diagnose of Freemartin Syndrome in Domestic Cattle (Bos taurus)

“…This results in an interchange of cells (hematopoietic chimerism) and plasma substances such as hormones between the twin embryos, leading to an intersexual state in the female (Harvey, 1976;Gustavsson and Johansson, 1980;Long, 1990). This intersexual condition is characterized by external genitalia that are essentially female in appearance, and internal genitalia that are affected to varying degrees, typically involving gonadal hypoplasia, inhibition of the Müllerian ducts, masculinization of the gonads, and stimulation of the Wolffian ducts (Tran et al, 1977;Edwards et al, 1994;Harikae et al, 2012).…”

A Clinical Case of an <b><i>SRY</i></b>-Positive Intersex/Hermaphrodite Holstein Cattle

“…containing gene 9 (SOX9) , anti-Müllerian hormone (AMH) and glial cell line-derived neurotrophic factor (GDNF) in the Sertoli cells to differentiate the bipotential gonads into testes [Harikae et al, 2012]. Without SRY , XX gonads normally develop into ovaries composed of granulosa and theca cells.…”

“…SOX9 factor, together with other proteins, is essential for both testicular differentiation and maintenance. In addition, gain or loss of function of SOX9 leads to XX and XY sex reversal [Harikae et al, 2012]. In farm animals, female pseudohermaphroditism is less common than male pseudohermaphroditism [Moriyama et al, 2010]; in fact, there are few reports of female pseudohermaphrodite cattle with normal karyotype (60,XX) [Burton and Momont, 1986;Takagi et al, 2005].…”