Intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa

ELSEVIER

INTRACYTOPLASMIC SPERM INJECTION OF IN VITRO MATURED OOCYTES OF DOMESTIC CATS WITH FROZEN-THAWED EPIDIDYMAL SPERMATOZOA L. Bogliolo, Ia G. Leoni, * S. Ledda, * S. Naitana, * M. Zedda, ’ A. Carluccio ’ and S. Pau’ ‘Obstetrics Section of the Institute of General Pathology Pathological Anatomy and Veterinary Obstetrics-Surgery Clinic 2Department of Animal Biology University of Sassari, Sassari, Italy Received for publication: Accepted:

January 4, 2001 May 15, 2001

ABSTRACT The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity. The first objective of this study was to compare the effect of two different culture media and two diITerent incubation times on in vitro maturation (IVM) of domestic cat oocytes. The second objective was to determine the developmental competence of in vitro matured cat oocytes after intracytoplasmic sperm injection (ICSI) with cat spermatozoa. Oocytes recovered t?om ovaries of ovariectomized cats were cultured either in TCM 199 medium or in synthetic oviductal fluid (SOF), both of which were supplemented with cysteamine, BSA, FSH, LH. Nuclear maturation was assessed afler 24 h and 40 h of incubation. Results of IVM showed that the percentage of oocytes reaching MI1 after 24 h and 40 h of incubation were significantly higher (P
0093-691WOl/$-see front matter PII: SOO93-691X(01)00621-5

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INTRODUCTION Because most of the 36 species of wild cats are classified as threatened, vulnerable or endangered (35), developing assisted reproduction techniques including in vitro maturation (IVM), in vitro fertilization (IVF) and embryo culture could play an important role in managing captive and natural populations as well as in sustaining both genetic and global biodiversity (63). The abii to mature and then fertilize oocytes in vitro may provide one approach for rescuing genetic material from nondomestic feline species that die or undergo an ovariohysterectomy for medical reasons. The domestic cat is often used as a model for the successful development of techniques as shown by high rates of fertilization from IVIWIVF, morula and blastocyst formation (40,65) and in the bii of offspring afler transfer of IVMAVF derived embryos (40). However, current methods for inducing cat oocyte maturation in vitro are suboptimal and the results obtained are poor compared to those achieved routinely in other species, such as mice (47), rats (61), cows (1, 49) sheep (52) and pigs (36). In fact IVM rates in the cat are usually around 40 to 60%, lower than in most other animal IVM systems (>80%). In the domestic cat bovine albumen is important as a protein source for meiotic maturation (12, 25, 31). Exogenous gonadotropins promote (12, 26), while fetal calf serum (3 1, 66) and estrus cat serum (12) retard the maturation frequency. The time necessary for oocyte maturation in vitro in the cat is still an open question. Some authors (3 1,43) reported that full meiotic maturation occurred after 24 h of culture while others (12, 26) indicated the need of 40 to 48 h of culture to obtain the highest maturation frequency. It is diicult to achieve in nondomestic felids the kequency of fertilization and cleavage similar to that reported in the domestic cat (5, 13,33,42). The main limiting factor appears to be the smalI quantity and poor quality of semen, which are characteristic of several nondomestic feline species (64). Micromanipulation-assisted fertilization, especially intracytoplasmic sperm injection (ICSI), could be used as an alternative to IVF to increase in vitro embryo production in nondomestic felids. This procedure, widely applied in the treatment of infertility in humans (9,37, 59), has been used in other species to increase embryo production in vitro and also to study Rmdamental aspects of gamete interactions during fertilization (32, 57, 58). In vivo viabiity of ICSI-produced embryos has been demonstrated in several domestic and laboratory species (3, 16, 28). In the Felidae family, kittens have been born following the transfer of cat embryos produced by ICSI of oocytes matured in vivo (41). Moreover, it has been shown that fertilization frequency and blastocyst development were similar after ICSI and IVF of oocytes recovered from gonadotrophin-treated cats (41). This (SOF) and investigate epididymal

study had two main objectives: to compare the effect of a simple culture medium a complex medium (TCM 199) on in vitro maturation of domestic cat oocytes, and to the developmental capacity of in vitro matured oocytes atler ICSI with frozen-thawed spermatozoa.

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MATERIALS AND METHODS Chemicals All chemicals in this study were purchased from Sigma Chemical Company (St. Lou&MO) unless stated otherwise. Oocyte recovery Domestic cat ovaries were recovered from cats subjected to ovariectomy or ovariohysterectomy at the veterinary clinic. Cats were anesthetized with tiletamine and zolazepam HCl (Zoletil 20, Virbac, Mii Italy, 10 mg/kg body weight, irn) and ovaries were excised aseptically via midventral laparotomy. The ovaries were immediately placed in phosphate-buffered saline (PBS) containing penicillin-G potassium (lOOpg/mL) and streptomycin sulphate (lOO&mL) at 37°C. Within 2 to 4 h of collection the ovaries were washed several times in fresh PBS and the oocytes released from the follicle by repeatedly puncturing the ovaries with 22-gauge needles until the tissue was minced. According to previously defined criteria (26), oocytes with a uniform dark, tinely granulated ooplasm that filled the zona pellucida and was completely surrounded by a tight corona radiata and cumuhrs cells, were selected for in vitro maturation. Experiment 1 The purpose of this experiment was to compare the effect of two different culture media and two different incubation times on in vitro maturation cat oocytes. Oocvte culture. Selected oocytes (n = 521) were randomly divided in two treatment groups for in vitro maturation: one containing medium TCM 199 supplemented with 0.4% BSA, cysteamine (100 mM), follicle stimulatmg hormone (FSH, 0.1 IU/mL, Pluset, Serono, Italy), luteinizing hormone (LH, 0.1 IU/mL, Phrset, Serono, Italy), and the other synthetic oviductal fluid (SOF, 56) supplemented with BSA, cysteamine, FSH and LH at the same concentration as in TCM 199. Oocytes were cultured in 2 mL of medium in groups of 50, in 35 mm Petri dishes at 39°C in a humidified environment of 5% CO2 in air. Assessment of nuclear maturation. Assessment of nuclear maturation was carried out after 24 h and 40 h of incubation. Cumuhts cells were removed by a combination of exposure to 0.2% (w/v) hyahrronidase for 1 to 5 min followed by mechanical displacement with a small-bore glass pipette. The denuded oocytes were then fixed in acetic-acid:ethanol (1:3) solution for 24 h, stained with 1% (w/v) aceto-lacmoid and morphologically evaluated for meiotic status. Experiment 2 The objective of this experiment was to determine the developmental competence of cat oocytes matured in vitro afler intracytoplasmic sperm injection of frozen-thawed epididymal spermatozoa.

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Semen collection. Spermatozoa were collected from the epididymides of cats following routine orchiectomy. Briefly, adult male cats were anesthetized with tiletamine and zolazepam HCL (Zoletil 20), and the epididymides with the vasa deferentia were removed with the testis through a scrotal incision. The spermatozoa were collected by flushing the cauda epididymidis through the ductus deferens with PBS by means of a 29-gauge needle, taking care to avoid blood contamination of the sperm suspension. Immediately after flushing, spermatozoa were frozen according the methods described by Hay and Goodrowe (20). Before ICSI, straws of frozen sperm were thawed by immersion in a 37°C water bath for 20 set and the thawed sperm was washed by centrifugation for 8 rnin at 350 x g in SOF with BSA, heparin (10 ug/mL) and hypotaurine (5 ug/mL). Intracvtonlasmic snerrn iniection (ICSI). For ICSI, 2 uL of the sperm suspension was added to a 10 uL droplet of 10% (w/v) polyvinylpyrrolidone in 0.9% NaCl in the center of a 60 mm culture dish lid. The ICSI was carried out afler 24 h of incubation in oocytes matured in vitro (n = 231) in SOF+BSA+FSH, LH, the culture medium in which we recorded the highest number of oocytes reaching MII. Matured oocytes were denuded of cumulus cells, as described above, and added to 40 uL drops (5 oocytes/droplet) of SOF with 0.6% BSA arranged in the manipulation chamber around the central drop. The dish was filled with sterile mineral oil. The ICSI was performed using a micromanipulator (Leitz Labovert,Wetzlar, Germany) under an inverted microscope (Labovert). Injection and holding pipettes were made using a micropipette puller (Model P-37, Sutter Instruments, Novado, CA, USA) and a microforge (D-900, Narishige Co. LTD, Setagaya-Ku, Tokyo, Japan). Sperm injection was carried out at room temperature. A single spermatozoon was aspirated tail tirst into the injection pipette (5 u id; 7 u o.d.). Intermittent aspiration and expiration was repeated until the spermatozoon was immobilized. An oocyte was held to the holding pipette (70 u i.d; 90 u o.d.) with the polar body at the 12 or the 6 o’clock position and the injection pipette at 3 o’clock position in order to avoid damage to the metaphase II spindle. After the tip of the injection pipette was inserted at the center of the oocyte, a small amount of ooplasm was aspirated to ensure oolemma rupture before sperm injection and then the spermatozoon was expelled with a minimum volume of accompanying medium. Intracytoplasmic sperm injection was also performed in a group of oocytes (n = 53) without a spermatozoon in the pipette (sham ICSI). At the end of injection a group of ICSI (n = 129) oocytes were activated for 5 min with 7% ethanol (ICSI activated oocytes).The re maining ICSI oocytes (n = 102) were not subjected to chemical activation (ICSI nonactivated). As a control for parthenogenetic activation MI1 oocytes (n = 92) were activated with ethanol (ACT). Culture in vitro followina ICSI. To assess developmental competence ICSI activated, ICSI nonactivated, sham ICSI and ACT oocytes were allocated (in groups of 20 to 25) to wells containing SOF with 0.6% BSA enriched with 2% MEM essential and 1% nonessential amino acids (AA) and incubated at 39°C under a 5% COz, 5% 02 and 90% Nz gas mixture for 7 days. At 28 h of in vitro culture (IVC) the number of cleaved embryos was recorded. Noncleaving oocytes were removed from the culture and stained with 1% aceto-lacmoid to determine meioticmitotic status, evidence of sperm or oocyte activation, or pronuclear formation. Day 3 and 5 embryos were transferred to fresh SOF+ BSA+ MEM essential and nonessential AA with

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5% fetal calf serum (FCS). On Day 7 of IVC the number of embryos developing to the blastocyst stage was visually determined and all embryos were stained with bisbenzimide (10 ug/mL,; Hoechst 33342) for the determination of the number of cells/embryo. Embryo development was classified according the following stages: 2 to 8 cells, 8 to 16 cells, morula (16 to >50 cells), blastocyst (> 50 cells with blastocelic cavity). Statistical Analysis The Chi-square test of independence (MINITAB release 12.1 for windows) was used to determine differences in the percentage of oocytes reaching MI1 (response variable) when affected by a categorical independent variable at 2 levels (maturation media and incubation time) and differences in the percentage of oocyte cleaving and reaching morula and blastocyst stages when affected by a categorical independent variable (chemical activation of oocytes after ICSI). Values were considered to be sign&ant at PcO.05. RESULTS Experiment 1 Results of in vitro maturation after 24 h of culture (Table 1) show that a signitkantly (P < 0.001) higher rate of oocytes reached MI1 (Figure 1A) after incubation with SOF (80%) than with TCM 199 (66.7%). Similar differences (P
129

31 (24.0)

10 (7.8)

86 (66.7)a

2 (1.5)

SOF

110

14 (12.7)

6 (5.5)

88 (80.0)b

2 (1.8)

abValues within columns with different superscripts differ (P
90

23 (25.6)

7 (7.8)

SOF

192

22 (11.5)

7 (3.6)

58 (64.4)a

2 (2.2)

4 (2.1) 159 (82.8)b abValues within columns with diierent superscripts differ (P
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Experiment 2. The results of developmental competence of ICSI activated oocytes and ACT oocytes are shown in Table 3. Cleavage frequency of ICSI activated oocytes afler 28 h of IVC was 82.2%. Alter 7 days of in vitro culture 26.4% and 32.1% of cleaved embryos were arrested at 2 to 8 (Figure 1B) and 8 to 16 cell stage respectively; 34.9% reached morula stage (Figure 1C) and 6.6 % developed to blastocyst (Figure 1D). Mean cell number of blastocysts is 91 f 26 (mean f SD). Eleven (47.8%) of the 23 oocytes that had not undergone the first division cleavage were at MI1 with a single polar body. Of these the spermatozoa was either not modiied (81.8%) or showed an enlarged sperm head (18.2%). The remaining noncleaved oocytes (12/23, 52.2%) extruded the second polar body and showed one female pronucleus and a not modified spermatozoon. As shown in Table 3, the cleavage rate of ACT oocytes was signiticantly lower than that of ICSI activated oocytes (40.2 vs 82.2%, P
Table 3. Cleavage after 28 hours of culture and in vitro development at Day 7 of ICSI activated cat oocytes and MI1 activated oocytes. Stage of development Oocytes n

Cleavage n (%)

2-8 cells n (%)

8-16 cells n (%)

Morula n (%)

Blastocysts n (%)

ICSI activated

129

106 (82.2)’

28 (26.4)

34 (32.1)

37 (34.9)

7 (6.6)

ACT

92

37 (40.2)b

24 (64.9)

13 (35.1)

0

0

(ACT: MI1 activated oocytes) &Values within columns with diierent superscripts diier (P
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Table 4. Cleavage after 28 hours of culture and development at Day 7 of ICSI nonactivated and sham ICSI cat oocytes. Stage of development Oocytes n

Cleavage n (%)

2-8 cells n (%)

8-16 cells n (%)

Morula n (%)

Blastocysts n (%)

ICSI Nonactivated

102

42 (41 .2)a

29 (69.0)

11 (26.2)

2 (4.8)

0

sham ICSI

53

8 (15.1)b

3 (37.5)

5 (62.5)

0

0

*Values within columns with different superscripts differ (P
DISCUSSION In this study we compared the effect on in vitro maturation of domestic cat oocytes of two different culture media: TCM 199 and SOF. The first medium is widely used for in vitro maturation of m oocytes of other species while the simple medium SOF, formulated for embryo culture in sheep and cattle (56), is used to improve development in bovine (8) and ovine embryos (61). More recently it has been tested for the in vitro maturation of dog oocytes (20). Our results showed that a greater proportion of oocytes cultured in SOF achieved complete nuclear maturation compared to those cultured in TCM 199. The percentages of MI1 oocytes recorded after maturation in the simple medium (80%) were higher than those described by other authors (12,23,3 1,41) and comparable to the maturation rate reported in livestock species such as cows and ewes (2,34). Percentages of MI1 oocytes observed after culture in TCM 199 were similar to those reported by other authors using the same culture system (40) or atter incubation with different complex media (14, 65). In contrast, a simple medium, mKRB, supplemented with BSA and FSH, was able to induce nuclear maturation only in a maximum of 55% of cat oocytes (12). The mKRl3 is composed of the same components as SOF, but their components have diEerent concentrations and this could explain the more successful results obtained in our experiment. An elegant study on the optimization of culture media for embryonic development in mice showed that wide differences in the abii to support development in vitro are due to the relative concentration of the components rather than to the presence or absence of a single component in the media (30). Analysis of the influence of different incubation times showed that full nuclear maturation is generally achieved within the tirst 24 h of culture in both media utilized. These findings disagree with previous results where a peak in MI1 oocytes was recorded at 30 h (26) or 40 to 48 h (12, 66), but they agree with other studies reporting that maturation can occur within 24 h of culture (3 1,40). The difference in maturation time may be related to the d&rent culture conditions used.

Theriogenology

Figure 1.

Developmental stages after ICSI and chemical activation of (A) in vitro matured domestic cat oocytes; (B) two cells embryo; (C) morula; (D) early blastocyst. Scale bar represents 30 pm.

The ICSI has been applied successfully in many mammalian species and offspring were reported afler transfer of ICSI embryos in humans (37), mice (28, 29), rabbits (22), cattle (16), sheep (3) and horses (52). Kittens have been born atler transfer to domestic cats of embryos produced by subzonal insemination (SUZI) and ICSI of oocytes recovered from treated donors (39,41). Pope et al. (41) reported 42.8% blastocyst formation after ICSI of cat oocytes matured in vivo using ejaculated cool-stored sperm. Other studies showed 15.4% (15) and 18.1% (3) development to blastocyst stage after ICSI of cat oocytes matured in vitro. Our results are lower than those reported by these authors (15, 38, 39). This could be due to the different embryo

Theriogenology

culture conditions used, such as culture medium, concentration of protein source and gas atmosphere,which can affect the embryo development. Oocyte sources could also intluence the different development to blastocyst. Several studies showed that compared to IVM counterparts (26,41,46,65), in vivo matured cat oocytes fertilize and develop well in culture (4553). In fact, it has been reported (38) that blastocyst cell numbers are lower for in vitro than in vivo matured oocytes after ICSI. Results of our experiment confirmed these data showing reduced blastocyst cell numbers of IVM oocytes fertilized by ICSI. An incomplete cytoplasmic maturation in our in vitro maturation system could have compromized the development of cat embryos to the blastocyst stage. It is known that cat oocyte nuclear and cytoplasmic maturation can be asynchronous (50) as has been established in other species (17, 18, 48). Cytoplasmic maturation is crucial for successful fertilization and embryo development (6, 7). In our research we also observed a high incidence of developmental block during the transition from morula to blastocyst. It has been reported that, under a variety of culture conditions, a large number of IVF-derived cat embryos develop in vitro to the morula stage, but most morulae fail to develop to blastocysts (23, 24,45, 53). In other species, in vitro developmental block has been overcome by modification of the energy substrates and other media constituents or by embryo co-culture with oviductal cells (11, 62). However, in the cat, changes in culture conditions such as protein source (23), media complexity (23) temperature and gas atmosphere (24) and cell co-culture techniques (53) did not improve the development to blastocyst. In other species, developmental block usually occurs at earlier embryo stages (8 to 16 cells) and is oflen associated with the maternal-zygote transition (MZT) in genome control (10, 55). In the cat, the MTZ occurs at the 5 to 8 cell stage during IVM/IVF embryo development (21) and consequently the MTZ is not directly related to the morula-to-blastocyst transition. Studies of in vivo embryogenesis (54) showed that, compared to other animals, in the cat, the transit from oviduct to uterus occurs at a advanced stage of development and coincides with the timing of in vivo embryo compaction and blastocele formation. It is possible that in the cat the transition between the oviductal and the uterine environments intluences the process of compaction and blastocyst formation and could suggest that the in vitro developmental block may be due to the lack of particular uterine factors necessary to the blastocyst formation. Our understanding of the requirements for embryo culture in the cat is very poor and we cannot exclude that suboptimal culture conditions are at least partially responsible of this developmental block in vitro. Investigations of in vitro embryo development requirements associated with the evaluation of in vivo developmental capacity of embryos produced by IVIWIVF may be useful to establish adequate culture conditions for embryo development to blastocyst. Our findings show that the activation of oocytes atler ICSI by ethanol treatment is necessary. In fact, we obtained a low cleavage frequency without the artificial activation after ICSI. The need to induce artiticial activation after ICSI is still an open question and mammalian oocytes respond differently to sperm injection. In the human (37), mouse (28) and rabbit (27), the injected sperm is able to restart the cell cycle of the oocyte, but this is not the case in several other species that require artilicial activation of the oocyte (4, 44). Other authors have reported that artiftcial stimuli are not necessary to achieve consistent fertilization/cleavage of cat oocytes matured in vivo (41). However, we can hypothesize that other factors, such as culture conditions and sperm preparation and quality, can intluence the early behavior of the oocyte after ICSI and the failure of fertilization could be related to the the inabiity to activate the molecular components involved in the second meiotic resumption.

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In conclusion, the results of our experiment demonstrated that in vitro meiotic maturation of domestic cat oocytes can be enhanced by culture with a simple medium supplemented with BSA and gonadotrophins. The data also showed that domestic cat embryos can be produced using ICSI from in vitro matured oocytes and frozen-thawed epididymal sperm. However, the evidence of the morula-to-blastocyst developmental block suggests that further studies are needed to improve the culture conditions for in vitro cat embryo development. This technology developed in the domestic cat could be successfully applied to rescue genetic material from sick or deceased nondomestic felids and when poor sperm quality hinders the use of in vitro fertilization protocol, and could therefore result in the ultimate preservation of rare or endangered cat species.

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35. Nowell

36.

37. 38.

39.

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