High‐purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells

Abstract

Mammalian spermatogenesis is still nowadays poorly understood at the molecular level. Testis cellular heterogeneity is a major drawback for spermatogenic gene expression studies, especially when research is focused on stages that are usually very short and poorly represented at the cellular level such as initial meiotic prophase I (i.e., leptotene [L] and zygotene [Z]). Presumably, genes whose products are involved in critical meiotic events such as alignment, pairing and recombination of homologous chromosomes are expressed during the short stages of early meiotic prophase. Aiming to characterize mammalian early meiotic gene expression, we have found the guinea pig (Cavia porcellus) as an especially attractive model. A detailed analysis of its first spermatogenic wave by flow cytometry (FCM) and optical microscopy showed that guinea pig testes exhibit a higher representation of early meiotic stages compared to other studied rodents, partly because of their longer span, and also as a result of the increased number of cells entering meiosis. Moreover, we have found that adult guinea pig testes exhibit a peculiar 4C DNA content profile, with a bimodal peak for L/Z and P spermatocytes that is absent in other rodents. Besides, we show that this unusual 4C peak allows the separation by FCM of highly pure L/Z spermatocyte populations aside from pachytene ones, even from adult individuals. To our knowledge, this is the first report on an accurate and suitable method for highly pure early meiotic prophase cell isolation from adult mammals, and thus sets an interesting approach for gene expression studies aiming at a deeper understanding of the molecular groundwork underlying male gamete production. ' International Society for Advancement of CytometryKey terms meiosis; spermatogenesis; flow cytometry; cell sorting; guinea pig; meiotic prophase; gene expression SPERMATOGENESIS can be viewed as the simultaneous and coordinated execution of three individual programs of gene expression: somatic proliferation of spermatogonia, meiosis, and spermiogenesis (1). In the seminiferous tubules of adult mammals, germ cells in different maturation steps coexist, with C (round spermatids, elongating and elongated spermatids, spermatozoa), 2C (several types of G1 spermatogonia, secondary spermatocytes) and 4C (different stages of primary spermatocytes, G2 spermatogonia) DNA content. Moreover, somatic Sertoli cells (2C) coexist with germ cells inside the tubules, which are surrounded by peritubular myoid cells and immersed in a stroma containing fibroblasts, lymphocytes, mastocytes, macrophages, and Leydig cells (all of them 2C).Beside the obvious importance of spermatogenesis as a source of male reproductive cells, along the lengthy first meiotic prophase significant events such as the recognition, alignment, pairing (synapsis), and recombination (crossing over) of homologous chromosomes take place (2-5). Particularly, meiotic homologous recombination has vital importance for genetic variability (6). Furthermore, mutations of

Induction of spermatogenesis after stem cell therapy of azoospermic guinea pigs

“…The duration of spermatogenesis in guinea pigs is approximately 34 days, with four cycles of 8.5 days (NOLLER et al, 1977). It has also been shown that the first spermatogenic wave lasts between 40 and 45 days in guinea pigs (RODRÍGUEZ-CASURIAGA et al, 2011). Therefore, 60 days (approximately 7 expected cycles) after cell transplantation, the animals were euthanized, and both testes were removed and transferred into 10% formalin buffer solution.…”

“…Hajihoseini et al: Azoospermia stem cell therapy wave in guinea pigs (RODRÍGUEZ-CASURIAGA et al, 2011). Although the seminiferous tubules of guinea pigs are highly enriched in meiotic cells as compared with mice and rats, there has been no study to evaluate the effects of stem cell therapy on the model of guinea pig azoospermia.…”

Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye

“…Besides, the inclusion of VDG afforded very interesting cytometric profiles with a significant number of cell populations distinguishable in the dot plots, even in species with no peculiar abundancy of certain stages such as L/Z in mouse [11]. Within 2C cells, different subpopulations can also be distinguished, but their identities are yet to be determined.…”

Revisiting testicular cell suspensions and meiocytes sorting

“…In this connection, we were the first to describe the presence of a bimodal DNA content peak in 4C cell populations, and to sort guinea pig L/Z and P MPI stages with minimal cross-contamination as shown by immunodetection using anti-SYCP3 antibodies (4). Recently, we have been able to achieve similar results analyzing mouse early meiocytes with Vybrant dye (Rodr ıguez-Casuriaga et al, Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye, Submitted).…”