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.

BackgroundIn Japan, Livestock Improvement Association of Japan started commercially producing sexed bovine semen 10 years ago, and sexed bovine semen is currently used for the artificial insemination (AI) in the farms. In this review, the authors introduce the technology for sperm sexing by flow cytometry, the efforts at commercializing sexed semen in Japan, and recent field data on artificial insemination of the cattle with sexed semen.MethodsIn the procedures of the flow cytometric method, X‐chromosome‐bearing sperm and Y‐chromosome‐bearing sperm were fluorescently stained, separated from each other by analyzing the difference in the DNA content, and cryopreserved. The authors surveyed the conception rates after AI with these sperm and sex ratios of the offspring with the cooperation from livestock associations, AI technicians, and farmers.Main findings (Results)Although AI with sexed semen was associated with lower conception rates in comparison with AI with conventional semen, the accuracy of sex selection using AI with sexed semen was beyond >90%.ConclusionSexed semen produced by flow cytometry has the potential to produce offspring of the preferred sex with high accuracy and reliability. Thus, it is expected that sexed semen is used for AI more frequently in the farms.

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The purposes of these studies were to examine the influence on the yield of sex-sorted sperm by the different size of sorting gate and to evaluate factors that affect the purity of sex-sorted sperm. As the sorting gate was expanded, so the yield of bovine sex-sorted sperm using flow cytometry was increased. At the same time, the purity of sex-sorted sperm became low. In addition, though the sorting gates were the same size, the purity of sex-sorted sperm differed among trials. These observations indicated the existence of factors that affect purity besides the size of sorting gate. To examine the yield of sex-sorted sperm, X-bearing sex-sorted sperm from 4 Holstein bulls were produced repeatedly 34 times by 3 flow cytometers. The sizes of sorting gates were fixed at 40–42%, 44%, and 46%. Each yield of sorting gate at 40–42%, at 44% and at 46% was compared. To evaluate factors that affect purity, X- or Y-bearing sex-sorted sperm were produced by one flow cytometer. These trials were repeated 160 times for the sorting of X-bearing sperm and 45 times for the sorting of Y-bearing sperm. Stepwise multiple regression analysis was used to analyse the relationships between the purity of sex-sorted sperm and the following sorting conditions, the percentage of oriented sperm, the percentage of dead sperm, degree of separation between X-bearing and Y-bearing sperm, the size of sorting gate, event rate, drop drive frequency, drop delay value and drop delay accuracy. The highest yield was acquired by sorting gate at 44%. The number of sex-sorted sperm was increased as sorting gate was expanded, however, the purity became low. The purities of the sperm by some trials using sorting gate at 46% were less than our acceptable lowest purity that was 90%. So that those sperm must be discarded. Therefore the yield of sorting gate at 44% was greater than sorting gate at 46%. Stepwise multiple regression analysis revealed that the factors for increasing purity of X-bearing sex-sorted sperm were the percentage of oriented sperm (P < 0.001), the degree of separation between X-bearing sperm and Y-bearing sperm (P < 0.001), the drop delay accuracy (P < 0.001), the event rate and the drop drive frequency, and the factor for decreasing purity of X-bearing sex-sorted sperm was the size of sorting gate (P < 0.001). On the other hand, the factors for increasing purity of Y-bearing sex-sorted sperm were the percentage of oriented sperm (P < 0.01), the degree of separation between X-bearing sperm and Y-bearing sperm (P < 0.01) and the event rate (P < 0.05), and the factor for decreasing purity of Y-bearing sex-sorted sperm was the size of sorting gate (P < 0.01). From these results, it can be concluded that the purity of sex-sorted sperm was not depend on simply by size of sorting gate but was more completely explained by other sorting conditions.