WO2011061677A1

WO2011061677A1 - Method to prolong and improve the functionality of spermatozoa in vitro

Info

Publication number: WO2011061677A1
Application number: PCT/IB2010/055190
Authority: WO
Inventors: Riccardo Calafiore; Carlo Foresta; Giovanni Luca; Daniela Zuccarello; Massimo Menegazzo; Andrea Garolla; Alberto Ferlin; Luca De Toni; Mario Calvitti; Ennio Becchetti; Francesca Mancuso; Claudio Nastruzzi
Original assignee: Università degli Studi di Perugia
Priority date: 2009-11-17
Filing date: 2010-11-16
Publication date: 2011-05-26
Also published as: ITRM20090595A1; IT1396725B1

Abstract

The present invention refers to a novel method for prolonging the viability of spermatozoa in vitro, comprising preparing co-cultures of spermatozoa and Sertoli cells, and to cell cultures obtained according to the method of the invention.

Description

METHOD TO PROLONG AND IMPROVE THE FUNCTIONALITY OF SPERMATOZOA IN VITRO

DESCRIPTION

The present invention refers to a novel method for prolonging the viability and/or improving the functionality of spermatozoa in vitro, comprising preparing co- cultures of spermatozoa and Sertoli cells, and to cell cultures obtained according to the method of the invention.

STATE OF THE PRIOR ART

Infertility is a problem which involves a high number of couples, estimated at over 6 millions in the U .S. alone. Infertility problems for over 30% of couples are caused exclusively by male infertility due to a reduced functionality of spermatozoa, such as, e.g., a reduction of sperm motility parameters (asthenozoospermia) or a low number of spermatozoa in the ejaculate (oligozoospermia). In order to be able to solve infertility problems various techniques of medically assisted procreation have been developed, such as, e.g., intrauterine insemination, in vitro fertilization, assisted fertilization and intracytoplasmatic sperm injection. All these techniques envisage the use of spermatozoa in vitro.

It is known that spermatozoa freshly ejaculated and placed in culture, though in cu bated i n specifi c med i a (l i ke , e . g . , th e BWW m ed i u m ) , ra pid ly an d progressively lose their functional features in terms of motility, viability and mitochondrial function (Calamela et al., Andrologia 33, 79-86, 2001 ). Calamera et al. (Andrologia 33, 79-86, 2001 ), demonstrate, in a very clear-cut way, that human spermatozoa cultures, such as those set up for medically assisted procreation methodologies, over +47 hours undergo a sharp loss of all functional parameters, such as motility, viability, DNA fragmentation and ability to perform acrosome reaction, negatively affecting the outcome of oocyte fertilization. The authors also show that said loss of viability and functionality can in no way be slowed down by in-culture addition of oxygen-free radical scavenger systems, such as catalase. Afterwards, Chi et al. (Human reproduction 23(5), 1023-1028, 2008) succeeded at improving the functional performance of human spermatozoa by adding to the system , besides catalase, also E DTA, on ly however not over +24 h after ejaculation. In the current state of scientific knowledge, there is no possibility to prolong the viability of spermatozoa cultures beyond +48 h keeping their functional features unaltered. The rapid and progressive loss of the features of viability and efficiency of spermatozoa in vitro negatively affects the outcome of techniques of medically assisted procreation , not allowing an accurate selection of the most viable spermatozoa or a n option al pre-treatment of the spermatozoa with substances improving the functional parameters, such as motility. Therefore, the need to propose novel methods for prolonging and/or improving the viability and functionality of spermatozoa in vitro was acutely felt in the state of the art.

SUMMARY OF THE INVENTION

It is therefore object of the present invention a novel in vitro method for prolonging the viability and/or improving the functionality of spermatozoa comprising the following steps:

a. incubating in a culture isolated Sertoli cells having a degree of purity of > 90% for a time comprised between 72 and 144 hours;

b. adding spermatozoa to said Sertoli cell culture;

c. maintaining said culture obtained at step b) in a Sertoli cell number/spermatozoa number ratio substantially comprised between 1 :5 and 1 :20 for a time comprised between 1 and 7 days.

The inventors highlighted, by measuring parameters such as motility, viability, mitochondrial functionality and oocyte penetration ability, that spermatozoa placed in co-culture with Sertoli cells according to the method of the invention exhibit higher values over time with respect to spermatozoa placed in culture according to the methods of the known art.

Therefore, the invention entails the following advantages with respect to the known art:

- prolonging viability times of spermatozoa in vitro allows to more accurately select the most functional ones, to be used immediately or upon pre-treatment with substances improving their functionality in techniques of medically assisted procreation.

- spermatozoa cultivated in vitro according to the method of the invention, besides remaining viable, motile and functional for over 7 days, do not incur capacitation nor premature acrosome reaction. These two processes can be activated at will, in vitro, when it will be decided to utilize spermatozoa to penetrate the oocyte by, e.g., one of the known techniques of medically assisted procreation.

- spermatozoa exhibiting a reduced fertility owing to a reduction of motility parameters can be cultivated in vitro according to the method of the invention, determining an improvement of motility parameters. Spermatozoa thus obtained could be used with greater probability of success for oocyte fertilization.

The present invention further refers to cell cultures comprising Sertoli cells and spermatozoa, obtainable with this method, and to their uses.

The advantages, features and application steps of the present invention will be made apparent in the following detailed description of some embodiments thereof, given by way of example and not for limitative purposes.

DETAILED DESCRIPTION OF THE FIGURES FIGURE 1 . Microphotographies of swine Sertoli cells. Figure 1A: Immunocytochemistry, obtained by incubating the preparation with anti-mullerian inhibiting factor antibodies (MIS). B: Immunocytochemistry, obtained by incubating the preparation with anti-vimentin antibodies. C-D: To demonstrate scarce presence of Leydig cells and peritubular cells, the preparation was subject to histochemical techniques to assess the presence of alkaline phosphatase, colouring with Fast- Red (C), and the activity of 3-3-hydroxy- steroidaldehydrogenase enzyme, colouring with Nitro-blue tetrazolium (D).

FI GU RE 2. A: Co-culture of human spermatozoa on Sertoli cell monolayer. B: human spermatozoa in control medium. The spermatozoa, clearly discernable from the underlying monolayer of Sertoli cells, remain viable and in motion throughout the observation period.

FIGURE 3. Results of functional tests (motility, viability and mitochondrial function) performed on spermatozoa in co-culture with SC. A-B: spermatozoa motility during co-culture remains constant, stabilizing at about 45 % (A+B+C motility) (Panel A) and 25 % (A+B motility) (Panel B) after 7 days of co-culture. C: Spermatozoa viability remains constant until day 4-5, then decreasing on day 7. D: Mitochondrial functionality, index of the energetic status of the spermatozoon, remains constant throughout the observation period. All experiments proved significant (p value) when compared to controls (Sertoli cell culture medium and BWW, the standard culture medium for spermatozoa).

FI GURE 4. Results of tests for the evaluation of intracellular calcium movement (capacitation ) (Panel A) an d acrosome reaction ( Pan el B) performed on spermatozoa in co-culture with SC. From the results it is deduced that spermatozoa after 7 days of co-culture with Sertoli cells incur no capacitation, nor acrosome reaction, thereby maintaining their fertilizing power intact.

FIGURE 5. Results of tests of acrosome reaction after induction with progesterone (Panel A) and of hamster egg penetration test, HEPT (Panel B). Further confirming the good quality of the spermatozoa at +7 days of co-culture and their fertilizing ability, is that when subject to induction of acrosome reaction they react as fresh spermatozoa. Moreover, subject to HEPT, they maintain their ability to penetrate the oocyte. DETAILED DESCRIPTION OF THE INVENTION

Glossary

Within the scope of the present invention the following definitions will be used with the following meanings:

Sertoli cells or SC: cells located in the wall of seminiferous tubules, having a trophic and immunomodulatory role on maturing germ cells;

spermatozoon (sperm): the germ cell or male gamete having the role of reaching the female gamete, the oocyte, to fertilize it during sexual reproduction;

asthenozoospermia: a reduction of sperm motility parameters; < 50% nemasperms with progressive motility;

oligozoospermia: reduction of number of spermatozoa in the ejaculate; < 20 million sperm/ml);

theratozoospermia: a presence in the ejaculate of < 30% spermatozoa having normal morphology;

medically assisted procreation: the whole of medical and biological techniques allowing reproduction outside of natural processes.

capacitation process: process consisting in the removal of a glycoprotein called acrosome-stabilizing factor from the outermost membrane of a spermatozoon (sperm). Capacitation initiates sperm activation processes, allowing encounter and contact with the oocyte.

acrosome reaction: during fertilization, union of the spermatozoon (sperm) with the secondary oocyte triggers the release of hydrolytic enzymes in charge of digesting the extracellular membrane of the oocyte, thereby facilitating the spermatozoon access to the plasma membrane of the same oocyte. This process is referred to as "acrosome reaction".

The method object of the invention comprises essential features that will hereinafter be described in more detail for possible embodiments for each of them. The first step provides the incubation of isolated Sertoli cells (SC) having a degree of purity of about >90% for a time period of at least 72 h.

Isolated SC may be obtained by various techniques known in the prior art. SC can be isolated from several animal sources, such as, e.g., swine, rat, mouse, dog, cat or human , preferably from prepubescent individuals. Animals, dead or after anaesthesia, are subject to bilateral orchiectomy. After removal of the epididymis, the testicles are su bject to multienzymatic d igestion . Once the digestion is complete, the tubular tissue is subject to filtration and the tubules thus obtained are placed in culture. In the prior art, various culture media are known which can be used both in steps of isolating SC and for maintaining them in culture, such as, e.g., the DMEM (or HAM-F12) medium.

In the present invention, isolated SC having a purity of about >90% are used. The degree of pu rity of Sertoli cells isolated and transferred in culture will mainly depend on the presence of other cell types, such as Leydig or peritubular cells, co- purified during the isolation process. The purity of Sertoli cells isolated and transferred in culture can be assessed with various methodologies known in the prior art: for i nstance, by im m u nocytoch emistry tech n iq ues , i n wh ich the preparation is incubated with anti-mullerian inhibiting factor (MIS), and fluorescein anti-vimentin antibodies, which respectively mark the MIS and vimentin molecules, both expressed only by the SC, and histochemical techniques for assessing both the presence of alkal ine phosphatase (colou ring with Fast-Red), typical of peritubular cells, and the activity of 3-3-hydroxy-steroidodehydrogenase enzyme (colouring with Nitro-blue tetrazolium) which on the other hand is typical of Leydig cells.

The authors of the invention highlighted that Sertoli cells with this degree of purity and a possible presence of Leydig cells in rates no higher than 5-8%, with respect to the number of total cells, give an improved functionality with respect to cells with a lesser degree of purity or an absence of Leydig cells.

I ncu bation of SC in cu ltu re could be performed at a temperatu re comprised between 32 and 37° C, preferably 37°C, under an atmosphere with about 5% C0₂ in standard incubators known to the man skilled in the art. The cell concentration in which cells are to be incubated could be substantially comprised between standard values, such as, e.g., 200x10³ and 270 x 10³ cells/cm². After a certain incubation time, the SC start adhering to the culture plates, forming a cellular monolayer to which spermatozoa are added as described in detail below. The incubation time before addition of spermatozoa is of at least 72 h, preferably of 96 h and anyhow no higher than 144 h.

In the second step of the method, spermatozoa are added to the SC culture obtained at the first step.

The spermatozoa could be added directly after ejaculation to said cell culture, or following a pretreatment aimed at obtaining spermatozoa with a greater degree of purity. The man skilled in the art, by using knowledge known in the state of the art, wi l l be ca pa bl e of sel ecti n g with out i nventive activity th e m ost s u itable pretreatment. For instance, in freshly ejaculated samples, using as pretreatment the Ficoll gradient selection method as described in the examples, spermatozoa could be mildly selected to remove the non-gamete cellular component. In an advantageous embodiment of the invention, spermatozoa are added to the SC culture so as to obtain a SC/spermatozoa final ratio comprised between about 1 :5 and about 1 :20.

An example of concentration of Sertoli cells to which to add spermatozoa is about 10 millions/ml; the man skilled in the art, on the basis of the present description and the teachings of the known art, will be capable of defining a suitable concentration. In an advantageous embodiment of the invention, before adding the spermatozoa, the Sertoli cells obtained at the preceding step are treated with a lysis buffer, like e.g. a Tris buffer, that allows eliminating the residual germ cells through osmotic lysis.

In the third step of the method, the cell culture comprising spermatozoa and Sertoli cells obtained at the second step is incubated, maintaining a SC number/spermatozoa number ratio substantially comprised between 1 :5 and 1 :20 for a time comprised between 1 and 7 days.

The cell culture comprising Sertoli cells and spermatozoa could be incubated at a temperature comprised between 35-38°C, preferably 37°C, under an atmosphere with about 5% C0₂ in standard incubators for a time comprised between 1 and 7 days. To ensure that spermatozoa viability remains high even at one week from the time at which spermatozoa are added in the culture, the SC/spermatozoa number ratio should be comprised between 1 :5 and 1 :20, preferably of 1 :10.

Substances suitable for cell culture optimisation, such as, e.g., enriched culture media, non-essential amino acids, relaxin, carnitine, vitamin D, antioxidants, could be added to the cell cultures obtained at the various steps of the method object of the invention; the man skilled in the art, on the basis of the known art, will be capable of selecting which compounds to add, and in what amount to add them.

As demonstrated and described in detail in the examples by functional tests, spermatozoa isolated from the co-culture obtained according to the method of the invention maintain high viability and functionality values after more than one week from their being added in culture with Sertoli cells.

The cell cultures of the invention could then be used to select the spermatozoa exhibiting best viability and functionality parameters, which therefore will have higher probabilities to fertilize the oocyte when used in techniques of medically assisted procreation.

In an embodiment, the method of the invention could therefore comprise a fourth step, in which one or more spermatozoa are isolated from the co-culture obtained at the third step of the method of the present description. Spermatozoa selection from the cell cultures object of the invention could occur by procedures known to the man skilled in the art.

The invention of the present description also refers to cell cultures comprising spermatozoa and Sertoli cells obtainable according to methods described herein and to the spermatozoa isolated from said cultures.

Object of the present invention are also compositions for prolonging the viability and/or improving the functionality of spermatozoa in vitro comprising Sertoli cells having a degree of purity of > 90% and cell culture media.

EXAMPLES

Example 1: Isolation of SC from prepuberal baby swine and culture preparation.

The SC were isolated from testicles of baby swine (7-15 days old) "Large-White". After anaesthesia through the i.m. administration of 0.1 mg/kg azaperon (Stresnil® 40 mg/ml, Janssen, Beerse, Belgium) and 15 mg/kg ketamine (Imalgene® 100 mg/ml, Gellini Farmaceutici), the swine were subject to bilateral orchiectomy. After the removal of the epididymis, the testicles are decorticated from the albuginea, finely chopped into small tissue fragments (1 -3 mm³) and immediately subject to a first enzymatic digestion based on collagenase P (Roche Diagnostics, S.p.A. , Monza, Italy) (2 mg/ml) in H BSS (Sigma Chemical Co, St. Louis, USA). The digestion is continued up to the separation of the seminiferous tubules. The collected tubules are then washed in HBSS and centrifuged at 500 rpm. After the wash, the tubules are incubated with a solution of HBSS containing trypsin (2 mg/ml) and DNAse I (Sigma). After the completion of the second digestion, the trypsin solution is diluted 1 :1 with Hank's + 20% FBS to stop the enzymatic activity thereof. After further washes with HBSS, the tubules are separated from the peritubular cells through a light centrifugation at 300 rpm. The "pellet" containing the tubular tissue is suitably filtered with a stainless steel filter with a 500 μηη mesh opening. Finally, in order to remove any peritubular and Leydig cells contaminating the preparation, the suspension containing the tubules is further centrifuged at 800 rpm for 5 min and the resulting pellet is treated for 7 min with a glycine 1 M solution and EDTA 2 mM in HBSS at pH 7.2.

The th us obtained tu bu les are placed in a cu lture in HAM F 12 (Eu roclone) supplemented with retinoic acid 0.166 nM (Sigma) and with 5 ml/500 ml insulin- transferrin-selenium (ITS) (Becton Dickinson#354352), at 37°C under an atmosphere with 5% C0₂. After 48 hours of culture, the SC start adhering to the culture plates, forming a cellular monolayer. In order to remove the residual germ cells, the SC monolayer is treated with a buffer, tris-(hydroxymethyl)-aminomethane hydrochloride (TRIS) (Sigma), that allows eliminating the residual germ cells through osmotic lysis. Finally, the SC are grown in the above conditions in polystyrene flasks. The SC obtained were analysed in terms of purity, viability and functionality.

The purity of the SC, which was higher than 90%, was assessed by immunocytochemistry techniques, incubating the preparation with anti-mullerian inhibiting factor (MIS), and fluorescein anti-vimentin antibodies, which respectively mark the MIS and vimentin molecules, both expressed by the SC only (Fig. 1 A, B). To prove the reduced presence of Leydig and peritu bu lar cells as possible contaminants, the SC preparations were subject to histochemical assessments. These tests allow assessing both the presence of alkaline phosphatase (colouring with Fast-Red), typical of peritubular cells, and the activity of the 3-3-hydroxy- steroidodehydrogenase enzyme (colouring with Nitro-blue tetrazolium) which on the other hand is typical of Leydig cells (Fig. 1 C, D). The results obtained with these histochemical assays have allowed to prove the presence of 5-8% of peritubular and Leydig cells; these cellular populations, moreover, are useful (when present in these proportions) to ensure a molecular "cross-talk" favourable for the correct functionality of these populations of testicular cells.

The viability of SC was determined by treatment with ethidium bromide (EB) and fluorescein-diacetate (FDA) (Sigma). The cells, observed by a fluorescence microscope, in all conditions showed a viability of > 95%.

The cell viability test is routinely conducted immediately after the isolation and on the second day of culture.

Example 2 Co-culture of human spermatozoa and swine SC.

Ejacu lated spermatozoa are obtai ned from patient after 3 days of sexual abstinence. After having waited for sample liquefaction, the spermatozoa are mildly selected to remove the non-gamete cellular component by 50%-75%-90% gradient method (Ficoll-Paque Plus [GE Healthcare] centrifuged at 800 rpm for 20 min at room temperature). Subsequently, the pellet of spermatozoa is washed with sperm washing medium [Irvine Scientific], placed in co-culture on plates containing the swine SC monolayer at the concentration of 10 millions/ml, and supplemented with the SC-specific culture medium described above (point A). The plates thus obtained are incubated at 37°C in an atmosphere enriched with 5% C0₂ (Figure 2). Example 3 Assessment of viability and functionality of spermatozoa in co-culture with Sertoli cells.

Every 2 days (up to a maximum of 7 days) functional tests were conducted on spermatozoa placed in culture with the Sertoli cells. The tests comprise assessment of nemasperm motility (automatic scanner), viability (eosin test), mitochondrial functionality (JC-1 ), DNA fragmentation (Tunel test), intracellular calcium (calcium orange), acrosome reaction (CD-46), penetration ability (Hamster egg penetration test). These tests highlighted that, in comparison with spermatozoa placed in the sole culture medium without SC, after 7 days:

- All functional parameters assessed are significantly more conserved with respect to the control (Figure 3);

The spermatozoa do not incur capacitation, nor premature acrosome reaction, as highlighted by tests assessing intracellular calcium and CD-46 exposure (Figure 4);

- The spermatozoa maintain the ability to penetrate the oocyte (Figure 5).

Hereinafter, the procedures used to perform the functional tests are reported in detail:

Example 4 Test for assessing exposure of phosphatidyl-serine by Annexin V-FITC, Apoptosis Detection Kit (BP Biosciences)

Test for assessing exposure of phosphatidyl-serine, a specific ligand of Annexin V, outside of the cell membrane, as a phenomenon characteristic of apoptosis in its early stages. Annexin V is used, conjugated to FITC (a fluorochrome having the property of emitting fluorescence in the green ), with a DNA-intercalating cell viability indicator such as propidium iodide (a fluorochrome emitting fluorescence in the red). I n short, seminal fluid (at least 2 million spermatozoa) is placed in an Eppendorf tube ( 1 .5 m l) and washed with P BS 1 X at 2500rpm for 1 0 min . Subsequently, the cells are resuspended with 100 μΙ of 1 X Binding Buffer at a concentration of 1 -2 million cells/ml and 10 μΙ of Annexin V-FITC (kept in refrigerator at +4°C) is added, letting incubate in the dark for 15 min. Then, it is resuspended with 500 μΙ of Binding Buffer and 4 μΙ of P I a re added (fi nal concentration 25ug/ml). The sample is thus ready to be analysed at the cytofluorimeter.

Example 5 Test for assessing spermatozoa energy by Mitoprobe JC-1 Assay Kit (mitochondria apoptosis) INVITROGEN

Assesses the energy state of spermatozoa, determined on the basis of the mitochondrial membrane potential. Lipophilic cation 5, 5', 6, 6' tetrachloro-1 , 1 ', 3, 3' tetraethyl-benzimidazolcarbocyanide iodate (JC-1 ) is used. JC-1 is in monomer form when mitochondrial ΨΓΠ is depolarized, and, if excited at 488nm, emits in the green; on the contrary, it forms multimeric aggregations when mitochondrial ΨΓΠ is polarized, and, if excited (590 nm) emits in the red. Spermatozoa are aliquoted inside an Eppendorf tube (1 .5 ml) with a concentration of about 2 x 10⁶, and 1 ml PBS and 10 μΙ probe are added. The Eppendorf tube is wrapped both with parafilm and aluminium foil and left to incubate for 30 min at 37° C in the dark. Subsequently, it is centrifuged at 2500 rpm for 10 min; at the end of this, the pellet is washed with 1 ml 1 X PBS and centrifuged at 2500 rpm for 5 min. Finally, the pellet is resuspended with 500 μΙ of 1 X PBS and cells are examined through cytofluorimetry.

Example 6 Assessment of degree of DNA fragmentation - DEATH DETECTION KIT FLUORESCEIN (TUNED

Assesses the degree of fragmentation of DNA, still present in the nucleus, in sperm population (late phenomenon of apoptosis) by fluorescent labeling of the free 3'- OH ends with FITC (fluorescein). A contrast staining with DAPI (blue) is used, allowing highlighting the positive viable state of a cell. Bring to room temperature 4% Paraformaldehyde in PBS (stored at + 4°C). After washing the seminal fluid with 1 X PBS in 1 :2 volume, the same is centrifuged for at 2000 rpm 10 min, supernatant is eliminated, 3 ml 1 X PBS are added and centrifuging is repeated after having thoroughly ruptured the pellet with a pipette. Then, the supernatant is eliminated and 50-100μΙ of 1 X PBS are added. Then, the pellet is smeared on a glass slide. After having let it dry, the preparation is fixed for 60 min at room temperature in dark room with paraformaldehyde. To conclude, it is washed 2 min with 1 X PBS. Subsequently, 100 microliters of permeabilization solution (Triton, sigma) are placed on the glass slides and it is incubated 2 min in refrigerator, setting the glass slides laid down inside a box. It is washed twice with 1 X PBS contained in two different jars, so as to eliminate all the permeabilization solution. Then, 50 μΙ probe are added to the samples, at 37 °C in humidified atmosphere for 60 min, and subsequently it is washed twice with 1 X PBS, for 2 min each wash, and with 40 ml 1 X PBS + 3 μΙ DAPI for 40 s. Finally, 10 μΙ Antifade is added on humidified glass slide, which is covered with a 24x40 cover glass and observed by 100X fluorescence microscope in immersion.

Example 7 Assessment of spermatozoon degree of capacitation by ACROSOME REACTION

Assesses the presence of externalized cd46 as an index of occurred acrosome reaction of the spermatozoon. The seminal liquid is washed with culture medium and centrifuged at 2000 rpm for 10 min. The pellet is resuspended in 100 μΙ of anti-mouse IgG (vector +4°C) and left to incubate 30 min in the dark. Subsequently, two washes are carried out and 10 μΙ CD46-FITC (BD Pharmingen at +4°C) are added to the resuspended pellet. It is left to incu bate 30 mi n i n the dark. Washes are carried out again ; after resuspending in 0.5 ml PBS, 4 μΙ PI (25ug/ml) are added and sample reading on the cytofluori meter is carried out.

Example 8 Assessment on calcium presence in the spermatozoon - CALCIUM ORANGE Assesses the presence of calcium in the spermatozoon as an index of capacitation status thereof. The seminal liquid is washed with PBS (3 ml) and subsequently centrifuged at 2500 rpm for 10 min. The pellet is resuspended in 0.5 ml PBS, 5 ul Calcium Orange (10uM, Invitrogen Molecular Probes +4°C) are added and the whole is left 30' in the dark at room temperature. Then, 3ml PBS are added and it is centrifuged at 2500 rpm for 10 min. Finally, the pellet is resuspended in 100 μΙ PBS e and a glass slide is set up for fluorescence microscope reading in the rhodamine filter.

Example 9 Pretreatment of spermatozoa with Ficoll gradient selection method.

Ficoll is a colloidal silicon-based substance allowing to separate cells according to their density gradient. I n practice, Percoll solutions are stratified in a tube, at increasing concentrations, the seminal liquid is deposed on top of the tube and it is centrifuged for about 30 min. Thus, a separation will be obtained of the elements present in the seminal liquid, in connection with their density. At the bottom of the tu be we wi l l fi n d the m ore moti le spermatozoa , whereas the n on-gamete component will be found at upper portions thereof. The sediment thus obtained is then diluted and washed before being used for the co-culture.

BIBLIOGRAPHIC REFERENCES

1 . Calamera, J .C., Fernandez, P. J ., Buffone, M.G., Acosta, A. A. , Doncel, G.F.

Effects of long term in vitro incubation of human spermatozoa: functional parameters and catalase effect. Andrologia 33, 79-86, 2001 .

2. Chi H.J., Kim, J.H., Ryu, C.S., Lee, J.Y., Park, J.S., Chung, D.Y., Choi, S.Y., Ki m , M . H., Chun, E.K., Roh, S.I. Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Human reproduction 23(5),1023-1028, 2008.

Claims

1 . An in vitro method for prolonging the viability and/or improving the functionality of spermatozoa, comprising the following steps:

a. incubating in a culture isolated Sertoli cells for a time comprised between 72 e 144 hours having a degree of purity of > 90%;

b. adding spermatozoa to said Sertoli cell culture;

c . m a i nta i n i n g sa i d cu ltu re o btained from step b) in a Sertoli cell number/spermatozoa number ratio substantially comprised between 1 :5 e 1 :20 for a time comprised between 1 e 7 days.

2. The method according to claim 1 , wherein Leydig cells are present in said isolated Sertoli cells in a ratio substantially comprised between 5 and 8% with respect to the number of total cells.

3. The method according to claim 1 or 2, wherein said Sertoli cells prior to addition of spermatozoa are treated with a Tris buffer.

4. The method according to any one of the claims 1 -3, wherein said isolated Sertoli cells are incubated at a temperature comprised between 36 and 38 C° and under an atmosphere with about 5% C0₂.

5. The method according to any one of the claims 1 -4, wherein said isolated Sertoli cells are incubated at a concentration substantially comprised between 200 x 10³ and 270 x 10³ cells/cm².

6. The method according to any one of the claims 1 -5, wherein said isolated Sertoli cells are obtained from swine, rat, mouse, dog, cat, ovine, bovine or human tissue.

7. The method according to any one of the claims 1 -6, wherein said spermatozoa before being added are treated to remove the non-gamete cellular component.

8. The method according to claim 7, wherein said spermatozoa before being added are treated to remove the non-gamete cellular component with the Ficoll gradient selection method.

9. The method according to any one of the claims 1 -8, comprising a further step wherein from said cell culture comprising spermatozoa and Sertoli cells one or more spermatozoa are isolated.

10. A cell culture comprising Sertoli cells and spermatozoa obtainable according to the method of any one of claims 1 -9.

1 1 . Spermatozoa with improved functionality and viability obtainable according to the method of claim 10.

12. Use of Sertoli cells for prolonging the viability and/or improving the functionality of spermatozoa in vitro.

13. A composition for prolonging the viability and/or improving the functionality of spermatozoa in vitro comprising Sertoli cells having a degree of purity of > 90% and a cell culture medium.