Separation of ram spermatids by sedimentation at unit gravity

Loir, Maurice; Lanneau, M.

doi:10.1016/0014-4827(74)90345-0

Cited by 21 publications

(5 citation statements)

References 16 publications

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“…Primary spermatocytes and round spermatids were separated by velocity sedimentation at unit gravity and sedimentation velocities, and mean cell volumes were determined with a Coulter Counter by the method of Loir & Lanneau (1974). The theoretical relationship, log V= K+ 3/2 log S, between cell volume, V, and sedimentation velocity, S, was verified with the present material.…”

Section: Methodssupporting

confidence: 58%

Seasonal variations in the response of the testis and LH levels to hemicastration of adult rams

Reviers¹,

Loir²,

Pelletier³

1976

Reproduction

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The weight and histology of the testis and plasma LH levels were analysed after hemicastration of adult Ile-de-France rams in spring or in autumn. After hemicastration, the remaining testes were significantly heavier than those of entire animals measured at the same time of year. At 4 or 6 months after hemicastration performed in spring, the remaining testes were hypertrophied by nearly 40% as compared to the testes of entire sexually active animals, assessed in autumn. The variations of intertubular tissue volume, total seminiferous tubule length, stem cell stocks, daily production of round spermatids, and cellular volume of primary spermatocytes paralleled the variations in testis weight. The annual decrease of the area of the Sertoli cell nuclei and of the yield of meiosis and beginning of spermiogenesis during the non-breeding season was prevented by hemicastration performed in autumn. Plasma LH levels were consistently elevated till autumn after hemicastration performed in spring. A positive and significant correlation was observed between LH levels and yields of spermatogonial divisions.

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Section: Methodssupporting

confidence: 58%

Seasonal variations in the response of the testis and LH levels to hemicastration of adult rams

Reviers¹,

Loir²,

Pelletier³

1976

Reproduction

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“…The cells were separated into populations according to the method of Lam, Furrer & Bruce (1970) as used by Loir & Lanneau (1974). The sedimentation chamber was filled with 20 ml DMEM, 20 ml cell suspension, and 680 ml of a non-linear gradient of 0-5, 2 and 4 % BSA in DMEM.…”

Section: Separationmentioning

confidence: 99%

Steroid Release in Vitro by Two Luteal Cell Types in the Corpus Luteum of the Pregnant Sow

Lemon¹,

Loir²

1977

Journal of Endocrinology

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107

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Corpora lutea from sows at 30, 60 and 90 days of gestation were dissociated enzymically, and the components of the resulting cell suspension were separated by sedimentation at unit gravity. Two luteal cell populations of 30-50 micronm diameter and 15-20 micronm diameter were obtained and superfused for up to 18 h with Dulbecco's modified Eagle medium, the cells being supported in a column in a matrix of Biogel. Fractions were collected every 30 min and assayed for progesterone and oestradiol-17beta. At 30 and 60 days of gestation the large luteal cells produced progesterone at an initial rate of approximately 100 ng/h/10(5) cells, which decreased to half this rate at 90 days. The smaller cells also released progesterone into the medium at approximately 15-20 ng/h/10(5) cells at all stages of gestation. At 30 days of gestation, neither cell type released significant amounts of oestradiol-17beta, but from 60 days onwards ,significant and increasing quantities were measured in the superfusates from the larger cells. Both cell types were perfused with porcine LH at the three stages of gestation, and both showed an immediate response in terms of progesterone release which decreased in magnitude with increasing age of gestation. The response of the smaller cells was greater than that of the larger cells.

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“…The isolation of homogeneous populations of the respective spermatogenic cells is an essential prerequisite for definitive biochemical studies of germ cell differentiation. The technique which has been applied to the adult testis most successfully for this purpose utilizes differential sedimentation velocity at unit gravity of cells which differ in volume (14,18,20,23,24,35). With the recent refinements introduced by Romrell et al (35), this technique provides purified populations of pachytene primary spermatocytes, round spermatids, and residual bodies, but does not yield germ cells at any step of development preceding the pachytene stage of meiotic prophase.…”

mentioning

confidence: 99%

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

Bellvé

Millette

et al. 1977

The Journal of Cell Biology

1,313

933

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A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes, and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by days 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear in increasing numbers between days 18 and 20. Cell separations were attempted throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity >99%) and primitive type A spermatogonia (90%); day 8, type A spermatogonia (91%) and type B spermatogonia (76%); day 18, preleptotene spermatocytes (93%), leptotene/zygotene spermatocytes (52%), and pachytene spermatocytes (89%).Mammalian spermatogenesis is a continuum of cellular differentiation in which three principal phases can be discerned: spermatogonial renewal and proliferation, meiosis, and spermiogenesis. The initial phase of spermatogonial proliferation occurs in the basal compartment of the seminiferous epithelium (7, 9) and consists of a mitotic proliferation of stem cells which form, in sequence, type A spermatogonia, intermediate spermatogonia, and type B spermatogonia (19,30,31,36). The type B spermatogonia divide to form preleptotene primary spermatocytes which undergo a final replication of nuclear DNA before entering meiotic prophase. The second phase, meiosis, occurs while the spermatocytes remain intercalated between cytoplasmic processes of adjacent Sertoli cells on the adluminai side of the intercellular Sertoli junctions. Meiotic prophase,

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Separation of ram spermatids by sedimentation at unit gravity

Cited by 21 publications

References 16 publications

Seasonal variations in the response of the testis and LH levels to hemicastration of adult rams

Seasonal variations in the response of the testis and LH levels to hemicastration of adult rams

Steroid Release in Vitro by Two Luteal Cell Types in the Corpus Luteum of the Pregnant Sow

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

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