1 Department of Medical Genetics, Faculty of Medicine, 2 Centre for Reproductive Genetics, Porto and 3 Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar, State University of Porto, Porto, Portugal
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Abstract |
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Key words: in-vitro maturation/meiosis/non-obstructive azoospermia/spermatogenesis
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Introduction |
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Both FSH and testosterone are needed to support germ cell differentiation through direct or indirect actions on Sertoli cells (Carreau, 1994; Gnessi et al., 1997
; Schlatt et al., 1997
). They enhance Sertoli cell responses to insulin. FSH and insulin then regulate the glucose metabolism and Sertoli cell lactate production, which is needed for normal spermiogenesis and spermatocyte RNA synthesis. FSH was suggested to play a determinant role in the survival of germ cells besides increasing spermatogonia proliferation (Hikim and Swerdloff, 1995
; Foresta et al., 1998
; Baarends and Grootegoed, 1999
), whereas testosterone, which is 50100 times more concentrated in the testes than in serum (Gunsalus et al., 1994
), has been implicated in spermatogonia and spermatocyte differentiation, in the conversion of round to elongated spermatids (Huang et al., 1987
; McLachlan et al., 1994
; O'Donnell et al., 1996
), and as an antiapoptotic substance on spermatocytes (Erkkila et al., 1997
; Print and Loveland, 2000
).
In the present study, human Sertoli and diploid germ cells were individually isolated and then co-cultured under different media conditions to study the role of FSH and testosterone in supporting germ cell survival in long-term in-vitro culture, resumption of meiosis and spermatid differentiation. Controls were compared with cases of meiosis arrest in order to determine if in-vitro culturing could overcome the meiotic arrest. The developmental potential of the in-vitro matured spermatids was also studied by microinjecting these cells into oocytes, with the resulting embryos being then analysed by fluorescent in-situ hybridization (FISH).
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Materials and methods |
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Male patients
Sixty-one patients with non-obstructive azoospermia and normal karyotypes participated in the present experiments that took place between November 10, 1998 and July 1, 2000. Patients referred for treatment came with a full urologist clinical evaluation, which included a diagnostic testicular biopsy. Recovery of male gametes for clinical treatment was performed by open testicular biopsy. After treatment, of the nine Sertoli cell only syndrome cases, none was confirmed as pure, as three showed one focus of spermiogenesis (presence of late spermatids/sperm) and six had one focus of meiosis arrest; of the 23 maturation arrest cases, eight had one focus of spermiogenesis (presence of late spermatids/sperm) and 15 were confirmed to be arrested at meiosis; and of the 29 cases with hypoplasia, mature sperm could be recovered in all patients.
Male germ cell isolation and culture
Each testicle biopsy was collected in sperm preparation medium (SPM; Medicult, Copenhagen, Denmark) and squeezed with surgical blades. The resultant fluid was diluted with SPM and washed by centrifuging at 500600 g, twice for 5 min each. When an excessive number of erythrocytes was present, the pellet was resuspended for 5 min in 2 ml of erythrocyte-lysing buffer (Verheyen et al., 1995), prepared with 155 mmol/l NH4Cl, 10 mmol/l KHCO3, and 2 mmol/l EDTA in water, pH 7.2 with KOH (all from Sigma, Barcelona, Spain, cell culture tested), and filtered by 0.2 µm (TPP, Switzerland). After washing, samples were digested (Crabbé et al., 1997
) for 1 h at 37°C, in a solution of SPM containing 25 µg/ml of crude DNase and 1000 U/ml of collagenase-IV (Sigma). After washing, the pellet was resuspended in IVF medium (Medicult) and incubated at 3032°C, 5% CO2 in air until use. A sample was then diluted in SPM, spread on a tissue culture plate and covered with light mineral oil (Medicult). Sertoli cells, elongated type-A spermatogonia, round spermatogonia and primary spermatocytes, secondary spermatocytes, and round spermatids were individually selected in an inverted Nikon microscope, equipped with Hoffman optics and a heated stage (32°C), using Narishige micromanipulators (Nikon, Tokyo, Japan) and micropipettes of 1520 µm in diameter (SweMed, Frolunda, Sweden). Selected cells were mixed in a culture drop containing 40 µl of Vero cell conditioned medium, with or without 25 U/l of recombinant (r)FSH (Serono, Geneve, Switzerland) and 1 µmol/l of testosterone (Sigma, water-soluble) prepared as described by Tesarik et al. (1998a), and cultured at 32°C with 5% CO2 in air, for up to 21 days. Vero cells (Vircell SL, Santa Fe, Granada, Spain) were prepared in IVF medium (BM1; Lab. Elliós, Paris, France) with 10% synthetic serum substitute (Irvine Scientific, CA, USA). The overlaying solution was taken for germ cell culture as conditioned medium, 2 days after monolayer formation (Cremades et al., 1999
).
Biochemical assays
In Vero cell conditioned medium, FSH concentrations were assayed by enzyme-linked immunosorbent assay (BioMérieux kit; Vidas Systems), and testosterone by radioimmunoassay (Tecam). Assays were repeated at three different experiments.
Microinjection experiments
Oocytes used for testing the developmental potential of in-vitro matured spermatids came from patients that had donated spare oocytes for research. Female patients with normal karyotypes elected for ICSI clinical treatment cycles were treated with a long gonadotrophin-releasing hormone analogue suppression protocol combining buserelin acetate (Suprefact; Hoechst, Frankfurt, Germany) with pure (p) FSH(Metrodin HP; Serono) or rFSH (Gonal F; Serono; or Puregon; Organon, Oss, The Netherlands). Ovulation was induced with human chorionic gonadotrophin (HCG, Pregnyl: Organon; or Profasi: Serono). Oocytes were recovered from large ovarian follicles by ultrasound-guided follicular aspiration, 36 h after HCG administration, using flush medium (Medicult) (Sousa et al., 1999). Oocytes were microinjected in SPM using the strong dislocation of the ooplasm (Tesarik and Sousa, 1995
), and then cultured for up to 6 days over Vero cell monolayers. Normal fertilization was assessed 1018 h after injection, and embryo cleavage and quality were evaluated 42 h later (Staessen et al., 1995
).
Fluorescent in situ hybridization
For FISH, a published technique (Coonen et al., 1994) was followed. Briefly, embryos were washed in Tyrode's salt solution (TSS; Sigma), and partially depellucidated in TSS containing 0.1 mg/ml of pronase (Merck, Darmstadt, Germany). After washings, they were transferred to 12 µl of lysis buffer (0.01 N HCl/0.1% Tween 20; Sigma) on poly-L-lysine (Sigma) coated slides. Isolated germ cells were prepared by direct transfer to lysis buffer. After nuclei isolation, slides were air dried, rinsed in water and phosphate-buffered saline (PBS; Sigma) for 5 min each, rapidly dehydrated in an ethanol series, and stored in dark conditions at 20°C until use. Slides were then incubated with pepsin (100 µg/ml; Sigma) for 20 min at 37°C, rinsed in water and PBS, fixed in 4% paraformaldehyde (Bio-Rad, Watford, Herts, UK) at 4°C for 10 min, rinsed in PBS and water, and dehydrated. The probe mixture, made of 6 µl of hybridization buffer (Vysis Inc., Downers Grove, IL, USA), 1 µl of each directly labelled
-satellite centromeric DNA probes and 1 µl of water, was applied to each slide under a coverslip. Probes used (Vysis) were CEP 18 (region 18p11.1q11.1, locus D18Z1; Spectrum Orange, 0.5 µl, and Spectrum Green, 0.5 µl), and CEP X (bands Xp11.1q11.1, locus DXZ1; Spectrum Green)/CEP Y (bands Yp11.1q11.1, locus DYZ3; Spectrum Orange).
Probes and nuclear DNA were denatured simultaneously at 75°C for 3 min, and then left to hybridize for 24 h at 37°C in a moist chamber. Post-hybridization washes consisted of 60% formamide (Fluka Chemika, Switzerland)/2x standard saline citrate (SSC)/0.05% Tween 20, at 42°C for 5 min, 2xSSC, for 5 min at 42°C, and 4xSSC/0.05% Tween 20, for 5 min at room temperature. After rinsing in water and PBS, slides were dehydrated, air-dried and mounted in 10 µl Vectashield antifade medium containing 1.5 µg/ml 4',6-diamidino-2-phenylindole (DAPI) to counterstain the nuclei (Vector Laboratories, Burlingame, USA). The efficiency of the FISH procedure was controlled using metaphase chromosomes and interphase nuclei from cultured lymphocytes. For this, peripheral blood cells from healthy male patients were stimulated with phytohaemagglutinin (Difco Laboratories, Detroit, USA) and cultured for 72 h at 37°C. A total of 100 lymphocytes were examined, and 95% of them gave positive signals for the expected number of copies. FISH images were recorded in a Nikon (Eclipse, E-400) epifluorescence microscope fitted with a CCD camera and appropriate software (Cytovision Ultra, Applied Imaging International, Sunderland, UK).
Statistical analysis
Where appropriate, the 2-test was used to evaluate the significance of difference between the percentages of two groups.
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Results |
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About 3080 Sertoli cells, 1030 elongated spermatogonia, and 200 primary spermatocytes and round spermatogonia were always used in each culture test (Figures 13). When available,
1020 secondary spermatocytes (Figure 4
) and a variable number of round spermatids (Figure 5
) were also added to the culture medium. In two control cases, round spermatids could not be found to be added to culture.
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In-vitro maturation in conditioned medium supplemented with rFSH
In controls (Table II), newly formed round spermatids were observed in only three cases (23.1%), and of all round spermatids, 46.2% remained arrested, 27.8% developed into abnormal elongating spermatids, and 26% grew a flagellum (100% of cases). Of the latter, 23.9% remained arrested, whereas 76.1% developed into normal elongating spermatids (84.6% of cases). About 88.9% of the normal elongating spermatids arrested in culture, and only 11.1% progressed to early elongated spermatids (15.4% of cases), of which all have remained arrested.
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In-vitro maturation in conditioned medium supplemented with rFSH and testosterone
In controls (Table III), round spermatids could be retrieved for culture in 87.5% of the cases, and newly formed round spermatids were observed in all cases. Of all round spermatids, 22.7% remained arrested, 38.8% developed into abnormal elongating spermatids, and 38.5% extruded a flagellum (75% of cases). Of the latter, 17.3% remained arrested and 82.7% developed into normal elongating spermatids (68.8% of cases). About 63.2% of the normal elongating spermatids then arrested in culture, whereas 36.8% progressed to early elongated spermatids (56.3% of cases). Of these, 37.1% arrested and 62.9% developed into late elongated spermatids (31.3% of cases), of which >80% matured into sperm, with most exhibiting an abnormal nuclear morphology.
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Comparison between the three different media
Comparisons within and between groups (Table IV) suggest that although meiosis induction is increased by FSH, significant differences only appear in the presence of FSH and testosterone. No major differences were found within each tested medium regarding the in-vitro maturation rate of round spermatids. On the contrary, significant differences were found between groups of different culture media, suggesting that FSH plays a role in the maturation to elongating spermatids (in controls), and that FSH plus testosterone induces significant increases of maturation in early and late spermiogenesis (in controls and case studies).
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Discussion |
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Because round spermatid injection proved to be of seldom beneficial interest, in-vitro culture of these cells has then been experimentally initiated. Using IVF medium, it was shown that 22% of round spermatids can grow flagella in
12 days, but these cells then became arrested and were incapable of inducing normal embryo development (Fishel et al., 1997
; Aslam and Fishel, 1998
; Balaban et al., 2000
). On the contrary, by using Vero cell monolayers, isolated round spermatids were shown to be able to mature into late spermatids and sperm in
712 days (Cremades et al., 1999
). More recently, co-cultures of Sertoli and diploid germ cells have shown that FSH is needed for stimulating spermatogenesis and testosterone for inhibiting Sertoli cell apoptosis (Tesarik et al., 1998a
,b
,2000a
,b
). With this method, differentiation of elongated spermatids from primary spermatocytes was reported to occur within 12 days, and was proven to be able to generate viable normal pregnancies (Tesarik et al., 1999
). However, this pace was high compared with that of the normal testicular cycle which needs more than 1 month to proceed through meiosis and spermiogenesis, or at least 16 days to develop from the late pachytene or secondary spermatocyte stages to elongated spermatids (Steele et al., 1999
).
To ascertain if the human spermatogenic cycle could be restored in vitro, we have developed an alternative protocol where, to avoid any possibility of contamination by a hidden focus of elongating or elongated spermatids, round germ cells and Sertoli cells were first individually isolated, and then mixed and co-cultured. Because in-vivo spermatogenesis needs a complex set of growth factors and interleukins (Kierszenbaum, 1994; Gnessi et al., 1997
; Schlatt et al., 1997
), we have used Vero cell conditioned medium, once it is known that these cells secrete several of those factors (Huang et al., 1997
; Desai and Goldfarb, 1998
). Comparison between different tested media suggests that FSH increases cell viability, besides stimulating meiosis and the early differentiation of spermatids up to the elongating stage, and that the association between FSH and testosterone further inhibits apoptosis, increases the meiotic index, and improves the maturation rate of all spermiogenic stages. These results confirm previous findings in rodents which have suggested that FSH plays a determinant role in the survival of the seminiferous epithelium and in spermatogonia proliferation (Huang et al., 1987
; Hikim and Swerdloff, 1995
; Foresta et al., 1998
; Baarends and Grootegoed, 1999
), but suggest primarily that it plays a role in the conversion of round to elongating spermatids. In relation to testosterone, our results confirm that it inhibits human Sertoli cell apoptosis (Tesarik et al., 1998a
) and increases the viability of human diploid germ cells (Erkkila et al., 1997
; Print and Loveland, 2000
). They also confirm previous data from rodents showing that testosterone induces spermatogonia and spermatocyte proliferation and acts as a crucial element in the conversion of round to elongated spermatids (Huang et al., 1987
; McLachlan et al., 1994
; O'Donnell et al., 1996
).
In-vivo spermatogenesis in the human has been studied by Heller and Clermont (1964). The complete cycle from dark spermatogonia to sperm took 90 days, and
74 days from pale spermatogonia (the mitotic product of dark spermatogonia) to sperm. Conversion of pale spermatogonia to primary spermatocytes through the intermediate production of spermatogonia B lasted for
26 days. Preleptotene spermatocytes then needed 16 days to develop to the late pachytene stage, and these entered meiosis and developed into round spermatids during the next 16 days. Finally, spermiogenesis took
16 days to attain the elongated spermatid stage and 23 more days to mature into sperm. Our experiments reproduced these physiological time delays, with round spermatids evolving into elongating spermatids in
35 days, into early elongated spermatids in
611 days, into late elongated spermatids in
812 days, and into sperm in
1016 days. Similarly, we observed that some primary spermatocytes (probably at pachytene) finished meiosis I in 23 days, and that secondary spermatocytes also took 23 days to finish meiosis II and give rise to early round spermatids.
Although cultures could remain viable for 23 weeks, most of the newly formed round spermatids must have originated from late pachytene spermatocytes and secondary spermatocytes, since most of them appeared in the first 34 days of culture. This is in accordance with previous rodent in-vitro studies, which have shown that the information needed for meiosis completion and to begin spermiogenesis is only translated at the late pachytene stage (Nakamura et al., 1978; Toppari and Parvinen, 1985
; Kierszenbaum, 1994
; Hue et al., 1998
). Nevertheless, the present data show that meiosis can be reinitiated in vitro at a rate of 57%, at least from samples containing late pachytene and secondary spermatocytes, and that normal late spermatids can differentiate at a rate of 1232% from round haploid cells. On the contrary, cells from patients with complete meiotic arrest showed a lower rate of meiosis reinitiation (3.3%), with only 5% of the newly formed round spermatids differentiating into late spermatids.
It remains to be explained how meiosis and spermatid differentiation could be resumed in cases of complete meiotic arrest. Two possible mechanisms were envisaged. First, secondary spermatocytes could have been misdiagnosed, and then those cases with a positive progression were in reality wrong diagnoses of meiosis arrest. Second, the mutation causing meiosis arrest could have happened de novo at an age when pachytene spermatocytes had already developed, or alternatively the mutation did not attain all foci of germ cells (Vogt, 1996; Page et al., 1999
).
The present experiments also revealed that the optimized conditions were not met, as some control cases were unable to progress through spermiogenesis. Possible causes could be the need for specific factors secreted by connective tissue cells that surround the seminiferous epithelium, the rupture of cell connections during cell dissociation, which are essential for sharing gene products such as mRNA encoded by sex chromosomes (Morales et al., 1998), the loss of the compartmentalization into apical and basal systems as determined by Sertoli cells in vivo, and the absence of renewal of the culture medium. However, the present investigation was conducted with isolated cells in microdrops in order to assure that no mature spermatids could be hidden in the tissue. Now that culture conditions and the pace of cell differentiation in vitro are better defined, culturing can be performed in tubes, avoiding cell dissociation and enabling the change of the upper part of the culture medium.
The developmental potential of the in-vitro matured spermatids, from either controls or case studies, was assessed by microinjection on spare mature oocytes donated for research. In-vitro matured round spermatids and normal elongating and elongated spermatids elicited a low fertilization rate (3138%), regular rates of blastocyst formation (28.6% with round cells and 46.2% with late spermatids), but most of the embryos displayed an abnormal sex chromosome constitution, except in the case of morulae from late spermatids. The low fertilization rate and the high abnormal genetic constitution of the embryos may not result from a deficient meiotic process in vitro but rather to the immaturity of the spermatid. Immaturity of the cytoplasm could cause improper oocyte activation, or the presence of two centrioles could induce abnormal chromosomal seggregation such as those found in haploid embryos derived from 2PN zygotes (Sousa et al., 1996,1998
; Tesarik et al., 1998c
). The oocyte activating competence was not found at the round spermatid stage in mice (Ogura et al., 1994
), but it was demonstrated in hamsters (Ogura et al., 1993
), rabbits (Sofikitis et al., 1996a
,b
) and bovines (Goto et al., 1996
). In humans, a normal activating competence was demonstrated at the round spermatid stage (Sousa et al., 1996
) and even at the secondary spermatocyte stage (Sofikitis et al., 1998b
). Similarly, nuclear immaturity could retard X chromosome decondensation, thus inducing aneuploidy through mitotic errors (Luetjens et al., 1999
). Although the culture system needs improvements, such problems may also be an inevitable consequence when using germ cells from non-obstructive azoospermic patients, which are supposed to have an intrinsic genetic problem, such as mutations affecting specific genes in the AZF region that are responsible for loss of spermatid differentiation (Vogt, 1996
), as also shown in rabbits with primary testicular damage (Sofikitis et al., 1996b
).
We have also tested abnormal (without flagellum) elongating spermatids, as they represented the majority of the in-vitro differentiated haploid cells, and late elongated spermatids with abnormal nuclear morphology, which represented the majority of the terminal in-vitro differentiated cells. As expected, due to the evident centriole abnormalities and deficient nuclear maturation, the fertilization rate was low (8.3 and 27.3% respectively), no blastocysts formed, and all embryos showed sex chromosome abnormalities.
In conclusion, the present experiments ensured long-term (23 weeks) in-vitro co-cultures of human Sertoli and diploid germ cells, enabling, at a physiological pace, some degree of spermatogonia proliferation, resume of meiosis of late primary and of secondary spermatocytes, and differentiation of round spermatids to elongated spermatids. However, if in-vitro differentiated normal late spermatids could originate blastocysts at a regular rate and with a normal chromosome constitution, they nevertheless displayed a low fertilization potential with most of the embryos showing developmental arrest and sex chromosome anomalies.
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Acknowledgements |
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Notes |
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References |
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Submitted on June 19, 2001; accepted on September 17, 2001.