Establishment of an organ culture system to induce Sertoli cell differentiation from undifferentiated mouse gonads

Hasegawa, Chinatsu; Yokoyama, Toshifumi; Umemura, Yuria; Kawanishi, Kohei; Miura, Yuuka; Takada, Nanako; Ohno, Shuji; Onaru, Kanoko; Omotehara, Takuya; Hirano, Tetsushi; Mantani, Yohei; Miki, Takanori; Hoshi, Nobuhiko

doi:10.1292/jvms.20-0036

Cited by 4 publications

(7 citation statements)

References 44 publications

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“…Agarose gel blocks of approximately 7 × 7 × 7 mm were prepared as described previously [ 7 , 9 ]. A groove within each block was fashioned using 19-gauge needle sections (Terumo, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…A groove within each block was fashioned using 19-gauge needle sections (Terumo, Tokyo, Japan). Cylindrical agar pieces were prepared as described elsewhere [ 7 ]. They were cut to the same length as the culture pieces.…”

Section: Methodsmentioning

confidence: 99%

“…On the other hand, the filter culture method did not maintain the gonadal morphology well. After culture, mouse gonads exhibited a flattened appearance, and the morphology of AMH-producing Sertoli cells in the testes underwent degradation [ 7 ]. In our previous study, we employed the commonly used agarose gel method in gonadal studies to investigate the direct diffusion pathway of AMH from the testis to the mesonephros under conditions closely resembling those found in vivo [ 9 ].…”

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confidence: 99%

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Establishment of an organ culture system to maintain the structure of mouse Müllerian ducts during development

KATO,

YOKOYAMA,

FUJIKAWA

et al. 2024

The Journal of Veterinary Medical Science

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We previously showed that the anti-Müllerian hormone (AMH), infiltrating from the testis to the mesonephros reaches the cranial and middle regions of the Müllerian duct (MD) and induces their regression using an organ culture in mice. However, it is difficult to maintain structural integrity, such as the length and diameter and normal direction of elongation of the caudal region of the MD, in conventional organ culture systems. Therefore, the pathway of AMH to the caudal MD region remains uncharted. In this study, we established an organ culture method that can maintain the morphology of the caudal region of the MD. The gonad–mesonephros complex, metanephros, and urinary bladder of mouse fetuses at 12.5 dpc attached to the body trunk were cultured on agarose gels for 72 hr. The cultured caudal region of the mesonephros was elongated along the body trunk, and the course of the mesonephros was maintained in many individuals. In males, mesenchymal cells aggregated around the MD after culture. Moreover, the male MD diameter was significantly smaller than the female. Based on these results, it was concluded that the development of the MD was maintained in the present organ culture system. Using this culture system, AMH infiltration to the caudal region of the MD can be examined without the influence of AMH in the blood. This culture system is useful for clarifying the regression mechanism of the caudal region of the MD.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Establishment of an organ culture system to maintain the structure of mouse Müllerian ducts during development

KATO,

YOKOYAMA,

FUJIKAWA

et al. 2024

The Journal of Veterinary Medical Science

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“…Both male and female ICR mice were purchased from SLC Japan (Hamamatsu, Japan) and maintained as described elsewhere. 32,51 Fetuses at 12.5, 13.5 and 15.5 dpc were collected immediately after euthanasia, which was accomplished under deep anesthesia with isoflurane. Noon of the day on which the mating plug was observed was designated as 0.5 dpc.…”

Section: Animalsmentioning

confidence: 99%

“…To clarify the pathway by which AMH diffused to each region, the gonad-mesonephros complexes at 12.5 dpc were prepared in five conditions (Figure 8A). Then, as described elsewhere, 32,51 each sample was placed in a groove of an agarose gel block created using the needle sections of 21-gauge needles (Terumo, Tokyo) and cultured for 72 h. A gel sheet (one of the small pieces from the agar sheet) of approximately the same length as the incision was inserted to prevent the tissue from reattaching (Figure 8A). Pieces of aluminum foil or vinyl, although impermeable, were not suitable for culture because they adhered to the tissue (data not shown).…”

Section: Organ Culturementioning

confidence: 99%

Direct diffusion of anti‐Müllerian hormone from both the cranial and caudal regions of the testis during early gonadal development in mice

Kato,

Yokoyama,

Okunishi

et al. 2023

Developmental Dynamics

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BackgroundThe Müllerian duct (MD), the primordium of the female reproductive tract, is also formed in males during the early stage of development, then regresses due to the anti‐Müllerian hormone (AMH) secreted from the testes. However, the detailed diffusion pathway of AMH remains unclear. We herein investigated the mechanism by which AMH reaches the middle region of the MD using an organ culture system.ResultsInjection of recombinant human AMH into the testis around the start of MD regression induced diffuse immunoreactivity in the mesonephros near the injection site. When the testis and mesonephros were cultured separately, the diameters of both cranial and middle MDs were significantly increased compared to the control. In the testis–mesonephros complex cultured by inhibiting the diffusion of AMH through the cranial region, the cranial MD diameter was significantly increased compared to the control, and there was no difference in middle MD diameter.ConclusionsThese results indicate that AMH, which infiltrates from the testis through the cranial region at physiological concentrations, induces regression of the cranial MD at the start of MD regression. They also indicate that AMH infiltrating through the caudal regions induces regression of the middle MD.

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Expression patterns of sex steroid receptors in developing mesonephros of the male mouse: three-dimensional analysis

et al. 2023

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The androgen pathway via androgen receptor (AR) has received the most attention for development of male reproductive tracts.The estrogen pathway through estrogen receptor (ESR1) is also a major contributor to rete testis and efferent duct formation, but the role of progesterone via progesterone receptor (PGR) has largely been overlooked. Expression patterns of these receptors in the mesonephric tubules (MTs) and Wol an duct (WD), which differentiate into the efferent ductules and epididymis, respectively, remain unclear because of the di culty in distinguishing each region of the tracts. This study investigated AR, ESR1, and PGR expressions in the murine mesonephros using three-dimensional (3-D) reconstruction. The receptors were localized in serial para n sections of the mouse testis and mesonephros by immunohistochemistry on embryonic days (E) 12.5, 15.5, and 18.5. Speci c regions of the developing MTs and WD were determined by 3-D reconstruction using Amira software. AR was found rst at the distal end (gonadal side) of MTs at E12.5, and the epithelial expression showed increasing strength from cranial to the caudal side. Epithelial expression of ESR1 was found in the cranial WD and MTs near the WD rst at E15.5. PGR was weakly positive only in the MTs and cranial WD starting on E15.5 but negative in the distal end of the MTs. This 3-D analysis suggests that gonadal androgen acts rst on the distal end of MTs but that estrogen is the rst to in uence MTs on the WD side, while potential PGR activity is delayed and limited to the epithelium.

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Establishment of an organ culture system to induce Sertoli cell differentiation from undifferentiated mouse gonads

Abstract: Running Head: CULTURE SYSTEM OF UNDIFFERENTIATED GONAD (40/40 characters) immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.

Cited by 4 publications

References 44 publications

Establishment of an organ culture system to maintain the structure of mouse Müllerian ducts during development

Establishment of an organ culture system to maintain the structure of mouse Müllerian ducts during development

Direct diffusion of anti‐Müllerian hormone from both the cranial and caudal regions of the testis during early gonadal development in mice

Expression patterns of sex steroid receptors in developing mesonephros of the male mouse: three-dimensional analysis

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