stimulation of in vitro oocyte ovulation by progesterone and homologous pituitary gonadotropic...

ISSN 1062�3604, Russian Journal of Developmental Biology, 2012, Vol. 43, No. 3, pp. 157–163. © Pleiades Publishing, Inc., 2012.Original Russian Text © M.N. Skoblina, B.F. Goncharov, 2012, published in Ontogenez, 2012, Vol. 43, No. 3, pp. 185–192.

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Maturation amphibian and fish oocytes exposed tohormones is well reproduced in vitro (Masui andClarke, 1979; Goetz, 1983; Goncharov et al., 1997).Many papers are dedicated to the study of mecha�nisms of the process of maturation (Nagahama andYamashita, 2008; D’Inca et al., 2010; Pang and Tho�mas, 2010; Sadler et al., 2010). It was far more diffi�cult to induce in vitro oocyte ovulation (Wallace andSelman, 1978; Goetz, 1983; Pinter and Thomas,1999; Patino et al., 2003); its mechanisms have beenstudied considerably worse, although certain successhas been achieved in this respect also (Patino et al.,2003; Lister and Van Der Krask, 2008; Sena and Liu,2008; Bobe et al., 2009; Dhillon and Liu, 2010).

The study of ovulation in sturgeons was actuallyimpossible until recently since researchers either failedaltogether to stimulate this process in vitro (Lutes,1985) or the percentage of ovulating oocytes was verylow, or the result considerably varied from experimentto experiment. The study of the effect of the composi�tion of the culture medium of ovarian follicles on theovulation of oocytes of stellate sturgeon demonstratedthat one of the factors affecting the result of hormonalstimulation in vitro is the medium osmolality. The per�centage of oocytes of stellate sturgeon ovulating invitro under the effect of an extract of sturgeon pituitar�ies or progesterone increased after dilution of the cul�ture medium (Goncharov et al., 2009). As initialmedia in this study, we used Ringer solution for cold�blooded animals modified for sturgeons (RMS)(Goncharov, 1978) and Leibovitz medium (L�15) with

the addition of sodium bicarbonate. The dilution ofthese media permitted obtaining a rather high andcomparable in the two media percent of ovulatingoocytes. The verification of their ability to develop�ment demonstrated that L�15 medium containing1.84 g/L of sodium bicarbonate and diluted to 69% isthe best. Nevertheless, in this medium, the ovulationof oocytes in vitro was obtained not in all females;therefore, it seemed expedient to continue to searchfor an optimal medium.

Previously Goncharov et al. (1997) demonstratedthat the percentage of oocytes maturing in vitro underthe effect of sturgeon pituitary extract increases withan increase in the osmolality of the culture medium. Itappeared that for the stimulation of maturation, mediawith an increased concentration of sodium bicarbon�ate are more efficient than media with a greater osmo�lality but with a smaller content of bicarbonate. As forthe effect of the concentration of sodium bicarbonateon the hormonal induction of ovulation, there wereonly some observations that urged us to study thisissue.

To obtain a high percentage of matured, ovulated,and capable of fertilization and development eggs, notonly conditions of incubation but also the use of opti�mal hormonal treatment are important. It is knownthat the ovulation of sturgeon oocytes is induced by agonadotropic pituitary hormone and progesterone.There are sufficient grounds to suggest that prostag�landins participate in the induction of their ovulation,as is shown for tailless anuran amphibians (Schuetz,

GAMETOGENESIS

Stimulation of in vitro Oocyte Ovulation by Progesteroneand Homologous Pituitary Gonadotropic Hormone in Sturgeons

M. N. Skoblina and B. F. GoncharovKoltsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119334 Russia

e�mail: [email protected] March 4, 2011; in final form, May 20, 2011

Abstract—We showed that the percentage of sturgeon oocytes ovulating in vitro in Ringer solution modifiedfor sturgeons (RMS) considerably depends on the concentration of sodium bicarbonate and the concentra�tion of progesterone. Under optimal conditions (0.5 g/L of sodium bicarbonate and 30 ng/mL of progester�one), it can be higher than 80. Oocytes that matured and ovulated under such conditions are capable of nor�mal development. In the best case, approximately 70% of developing embryos (of the number of ovulatedoocytes) reach the stage of hatching (dead�line of observation). This method of producing offspring based onthe insemination of oocytes that have matured and ovulated in vitro can be used in work with single femalesof rare and disappearing sturgeon species.

Keywords: sturgeons, oocyte, maturation, ovulation in vitro, culture medium, sodium bicarbonate, progest�erone, pituitary, gonadotropic hormone.

DOI: 10.1134/S1062360412030083

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SKOBLINA, GONCHAROV

1986; Chang et al., 1995; Sena and Liu, 2008), bonyfish (Goetz et al., 1989; Goetz and Garczynski, 1997;Lister and Van Der Kraak, 2008), and mammals(Hedin et al., 1987; Bridges et al., 2006; Kurusu et al.,2009).

A key enzyme transforming arachidonic acid intoprostaglandins is prostaglandin synthase or an induc�ible isoform of cyclooxygenase (COX�2) (Vane et al.,1998). It was shown that in the process of induction invitro of maturation and ovulation of Xenopus laevisoocytes with progesterone and human chorionicgonadotropin the expression of mRNA of COX�2increases (Sena and Liu, 2008). The data obtained onmammals indicate that the activity of this enzymedepends on the concentration of progesterone.Progesterone can both increase the formation of pros�taglandins (Hermenegildo et al., 2005; Bridges et al.,2006) and decrease it (Thorbun et al., 1973; Gleesonet al., 1974; Hellberg et al., 1996; Hedin and Eriksson,1997; Diaz et al., 2002).

In experiments on induction of maturation ofsturgeon oocytes, progesterone in a concentration of1 µg/mL is usually commonly used. Our preliminaryexperiments demonstrated that it at least by an orderof magnitude exceeds the maximum efficient, and theaforementioned published data indicate that such ahigh concentration via inhibition of formation of pros�taglandins can negatively affect the induction of ovu�lation. Thus, in this study, we investigated the effect oftwo factors that can affect the hormonal induction ofsturgeon oocyte ovulation of oocytes in vitro—theconcentration of sodium bicarbonate and the concen�tration of progesterone. To judge as to how normallythe processes of maturation and ovulation stimulatedin vitro take place, we inseminated mature eggs andobserved the course of their development in the major�ity of experiments. Because of the absence of sperm,insemination was not performed only in experimentslisted in Table 2.

MATERIAL AND METHODS

The study was performed from April to May. Themain objects were follicles of sterlet (Acipenser ruthe�nus L.). One experiment was conducted also on folli�cles of the hybrid between Russian sturgeon (Acipensergueldenstaedti Brandt) and Siberian sturgeon (Aci�penser baerii Brandt). Females of sterlet and the hybridwere cultivated at Krasnodar (Adygei) sturgeon fishfarm. Follicles were obtained from females of the ster�let and the hybrid that were at this moment in basinswith a water temperature of 10°С.

Ovarian follicles were extracted from the femalebody using a special metallic probe and were placedinto a 75% medium L�15 (~15°). They were repeat�edly washed off with the same medium and kept in it at4°С before the experiment from 3 to 4 h up to 24 h.Prior to distributing follicles over a Petri dish, theywere washed off with media corresponding to those in

which they were subsequently incubated. Penicillin(500 units/L) and streptomycin (0.25 g/L) were addedto the medium.

As inducers of maturation and ovulation ofoocytes, we used progesterone or preparation of stel�late sturgeon pituitary gonadotropic hormone(aciGTH) purified according to the modified methoddescribed previously (Burzawa�Gerard et al., 1975).Progesterone was introduced in a Petri dish as a con�centrated alcohol solution. Final hormone concentra�tion in the culture medium was 16 to 1000 ng/mL, andthe alcohol concentration was 0.05%; aciGTH wasintroduced as a concentrated solution prepared onRMS with 0.5 g/L of sodium bicarbonate. Final con�centration of aciGTH was 2 µg/mL.

Follicles were incubated in 5 mL or 7.5 mL of themedium in a ventilated plastic Petri dish with a diam�eter of 35 and 55 mm, respectively. Twenty to twentytwo or 30–33 follicles were placed in a dish, respec�tively. Incubation of follicles was performed in a ther�mostat at a temperature of 16 ± 0.2°C. Ovulation wasestimated by counting follicle envelopes that separatedfrom the oocyte.

Insemination was performed by the semidrymethod commonly used in sturgeon pisciculture(Dettlaff et al., 1993). Before insemination, RMS waspoured out, and water and sperm were added simulta�neously as 1 : 25. Sperm was preliminarily checked formobility.

To determine significance of differences in the per�centage of ovulated oocytes, chi�square test was usedfor qualitative indices.

RESULTS

The Effect of the Composition of the Culture Mediumon the Induction with Hormones in vitro of the Ovulation

of Sterlet Oocytes

Table 1 shows results of experiments on incubationof follicles of eight sterlet females in two differentmedia (RMS containing 0.5 and 2 g/L of sodiumbicarbonate) in the presence of two hormonal prepa�rations (progesterone and aciGTH). In this experi�ment, the efficiency of hormones in the used concen�trations in both media was approximately equal. It isseen that both progesterone and aciGTH in a mediumwith a decreased concentration of sodium bicarbonateonduce ovulation in a greater percentage of cases in allfemales. The effect of the concentration of sodiumbicarbonate was slightly greater with the use of proges�terone. Besides, it was recorded that with a decrease inthe concentration of sodium bicarbonate, not only thepercentage of ovulated oocytes increases but the pro�cess of ovulation starts earlier. Asterisks denote thelevel of significance of differences.


STIMULATION OF IN VITRO OOCYTE OVULATION BY PROGESTERONE 159

The effect of the Concentration of Progesterone on the Ovulation in vitro of Sterlet Oocytes

We used three concentrations in experiments. Thelowest (16 ng/mL) comprised approximately a half ofthat used in experiments described above, the average(128 ng/mL) was greater than it by a factor of fourapproximately, and the greatest (1 µg/mL) equaledthat used by us previously for induction of maturationand ovulation of oocytes. In this experiment, we usedRMS with 0.5 g/L of sodium bicarbonate as an incu�bation medium.

Experiments were conducted on follicles of foursterlet females. The results of experiments are pre�sented in Table 2.

It is seen that the percentage of ovulated oocytesdecreases with an increase in progesterone concentra�tion. The exception is made by only female 3, in whichthe percentage of oocytes that ovulated under theeffect of the first two concentrations is approximatelyequal. Ovulated oocytes had a good appearance and,upon transfer to water, activated and divided parthe�nogenetically.

Capacity for Development of Sterlet Oocytes Maturedand Ovulated in Different Media under the Effect

of Progesterone or Gonadotropin of Stellate Sturgeon

All ovulated oocytes of sterlet female 1–5 (Table 1)were inseminated. The percentage of normally devel�oping embryos was determined at stages 5 (4 blas�tomers), 17 (the end of gastrulation), and 36 (hatch�ing) (Detlaff et al., 1993). The percentage of normallydeveloping embryos was calculated from the totalnumber of follicles taken for the experiment and fromthe number of ovulated oocytes. Unfortunately, wefailed to observe the development of embryos in allfemales. The data obtained are listed in Table 3. Theyturned out to be not quite equal for different females.In female 1, a considerably higher percentage of nor�mal embryos was obtained after insemination ofoocytes that matured and ovulated in RMS with0.5 g/L as compared to 2 g/L of sodium bicarbonateindependently of the used hormonal preparation. Infemale 2, against the background of the low percent�age of ovulation in all variants of the experiment, agreat loss at all stages of development was observed.Only in one variant (induction of ovulation withprogesterone in RMS containing 0.5 g/L of sodiumbicarbonate) up to the stage of hatching, a small num�ber of embryos survived. In female 3, against the back�ground of a sufficiently high percentage of oocytes thatovulated under the effect of both progesterone andaciGTH (especially in RMS with 0.5 g/L of sodiumbicarbonate), only embryos that developed fromoocytes matured and ovulated under the effect ofprogesterone reached the stage of hatching. At folliclesof the last two females, the experiment was slightlyreduced according to the number of variants (three

Table 1. Effect of concentration of sodium bicarbonate inRMS on the ovulation of sterlet oocytes stimulated byprogesterone or aciGTH

Percentage of ovulated oocytes

Concentra�tion of sodium

bicarbonate (g/L)

0.5 2.0 0.5 2.0

Stimulator: progesterone 30 ng/mL aciGTH 2 µg/mL

Female 1 85(51)

18** (11)

73 (44)

22** (13)

Female 2 42(25)

14** (3)

32 (7)

18 (4)

Female 3 82(18)

50* (11)

73(16)

64 (14)

Female 4 64(21)

– 18 (6)

15(5)

Female 5 70(23)

– 64 (21)

21** (7)

Female 6 85(28)

– 76 (25)

30** (10)

Female 7 42(14)

0** (0)

73 (24)

9** (3)

Female 8 88(29)

9** (3)

73 (24)

18** (6)

Females 1–8 70 18 60 25

The number of ovulated oocytes is provided in parentheses.Differences between the number of oocytes ovulated in RMS with0.5 and 2 g/L of sodium bicarbonate are significant at a level:* <0.05, ** <0.005. In the absence of an asterisk, differences areinsignificant.Gaps in the table are related to the absence of a sufficient numberof follicles for performing this variant of the experiment.

Table 2. Effect of progesterone concentration on the ovula�tion in vitro of sterlet oocytes in RMS with 0.5 g/L of sodi�um bicarbonate

Percentage of ovulated oocytes

Concentration of progester�one, ng/mL

16 128 1000

Female 1 59 (13) 36 (8) 27 (6)

Female 2 82 (18) 32 (7) 18 (4)

Female 3 75 (15) 85 (17) 20 (4)

Female 4 64 (14) 32 (7) 14 (3)

Female 1–4 70 46 20

Number of follicles per dish is 20–22. In parentheses is the numberof ovulated oocytes.

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SKOBLINA, GONCHAROV

instead of four). Besides, according to technical rea�sons, the quality of the obtained ovicells was assessedonly in the very beginning of development. Towardsthis stage again the best result was obtained in a variantof experiment with the use of RMS containing 0.5 g/Lof sodium bicarbonate, and progesterone as a stimula�

tor (female 4). In female 5, in this medium the effi�ciency of both stimulators turned out to be equal.

In a single experiment performed on follicles of thehybrid between Russian sturgeon and Siberian stur�geon (Table 4), the variant in which RMS containing0.5 g/L of sodium bicarbonate and aciGTH as a stim�

Table 3. Development of sterlet embryos obtained after insemination of oocytes matured and ovulated in vitro under theeffect of different stimulators and in different media

Number of a female Stimulator

Concentra�tion of sodium bicarbonate,

g/L

Total number

of follicles

Number of ovulated

oocytes

Percentage* of normal embryos at the stage:

4 17 36

1 Prog. 0.5 60 51 88 (75) 86 (73) 69 (58)

Prog. 2 60 11 82 (15) 54 (10) 36 (7)

aciGTH 0.5 60 44 98 (72) 95 (70) 68 (50)

aciGTH 2 60 13 77 (17) 69 (15) 38 (8)

2 Prog. 0.5 60 25 72 (30) 64 (27) 36 (15)

Prog. 2 33 3 67 (6) 0 (0) 0 (0)

aciGTH 0.5 30 7 57 (13) 0 (0) 0 (0)

aciGTH 2 33 4 50 (6) 50 (6) 0 (0)

3 Prog. 0.5 22 18 100 (82) 89 (73) 56 (45)

Prog. 2 22 11 82 (41) 73 (36) 55 (27)

aciGTH 0.5 22 16 100 (73) 63 (45) 0 (0)

aciGTH 2 22 14 50 (32) 43 (28) 0 (0)

4 Prog. 0.5 30 21 90 (63) not defined not defined

aciGTH 0.5 30 6 67 (13) not defined not defined

aciGTH 2 33 5 100 (15) not defined not defined

5 Prog. 0.5 30 23 78 (60) not defined not defined

aciGTH 0.5 33 21 100 (64) not defined not defined

aciGTH 2 30 7 86 (20) not defined not defined

* With respect to the number of ovulated oocytes; in parentheses, with respect to the total number of oocytes in the given variant of theexperiment.

Table 4. Development of embryos of the hybrid between the Russian sturgeon and Siberian sturgeon ovulated in vitro underthe effect of different stimulators and in different media

Stimulator Concentration

of sodium bicarbonate, g/L

Total number of follicles

Number of ovulated

oocytes

Percentage of normal embryos at the stage:

4 17 36

Prog. 0.5 31 8 75 (19) 38 (10) 38 (10)

aciGTH 0.5 31 28 100 (90) 96 (87) 79 (71)

aciGTH 2 31 4 75 (10) 50 (6) 50 (6)



ulator were used appeared to be the best in the level ofovulation and the potential of development.

DISCUSSION

In the previous study (Goncharov et al., 2009) ded�icated to the elaboration of the method of hormonalinduction of ovulation in vitro we found that it is pos�sible to obtain a high percent of ovulated oocytes ofstellate sturgeon with a decrease in osmolality of thecultivation medium of follicles via dilution of RMS;however, under these conditions, the process of theirmaturation is disturbed. Such oocytes are incapable offertilization and/or development.

We proposed that a low percent of ovulated oocytesfrequently obtained in an undiluted RMS in vitro canbe connected with a high content of sodium bicarbon�ate in RMS rather than with osmolality. This supposi�tion was completely supported. The decrease in theconcentration of sodium bicarbonate from 2 to 0.5 g/Lled to a considerable increase in the percent of ovu�lated oocytes independently of the used stimulator(Table 1).

Thus far it is difficult to judge the mechanism of theeffect of sodium bicarbonate on ovulation. It was longago established that its concentration is an importantfactor affecting the maturation of sturgeon oocytes invitro. An increase in the concentration of bicarbonateto definite limits increases the sensitivity of follicles togonadotropic hormones of hypothysis (Goncharov,1978), but does not affect the maturation of oocytesinduced by progesterone (Goncharov et al., 1997). Inaddition, at a certain physiological state of follicles,their incubation in RMS containing an increased con�centration of sodium bicarbonate (2–3 g/L) inducesmaturation of oocytes in the absence of hormones.There are all grounds to assume that the observedeffects of an increased concentration of this salt aredetermined more likely by a specific intervention intosignal pathways providing some functions of cellsrather than by its contribution to the medium osmola�lity or the effect on the medium pH. It is known, forinstance, that bicarbonate ion activates the solubleadenylate cyclase in sperms (Garbers et al., 1982; Hesset al., 2005) and early embryos of mammals (Chenet al., 2010), and this activation is necessary for thecapacitation of sperms and the development ofembryos.

In this study, for the induction of maturation andovulation of oocytes in vitro, we used a considerablylower concentration of progesterone—30 ng/mLinstead of 1000 ng/L. Transition to a lower concentra�tion was based on a suggestion that in sturgeons, as inteleostean fish (Goetz et al., 1989; Goetz and Garc�zynski, 1997; Lister and Van Der Kraak, 2008) andamphibians (Schuetz, 1986; Chang et al., 1995; Senaand Liu, 2008), prostaglandins are involved in theinduction of ovulation, and high doses of progester�one, according to the cited previously published data,

obtained true on mammals can inhibit their produc�tion. The use of a low concentration of progesteroneactually yielded a good result (in six females of eightthe percent of ovulated oocytes was higher than 50)(Table 1), which testifies in favor of the made sugges�tion. At the present time, the suggestion about theinvolvement of prostaglandins in the stimulation ofovulation of sturgeon oocytes is verified.

Note that, in studies on stimulation in vitro of theovulation in some species of teleostean fish, relativelysmall concentrations of meiosis�inducing steroid wereefficient. For instance, for the Japanese eel (Anguillajaponica), this is 10–100 ng/mL (Kagawa et al., 2003),and that for Atlantic croaker (Micropogonies undula�tus) is 290 ng/mL (Yoshizaki et al., 2001; Patino et al.,2003).

To verify a possible inhibiting effect of big concen�trations of on ovulation, an experiment was performedon four sterlet females, and upon the use of 1 µg/mL,actually a considerably worse result was obtained ascompared with 128 and 16 ng/mL (Table 2).

Thus far it is difficult to judge the range of progest�erone concentrations optimal for induction of ovula�tion. It is highly apparent that it will differ in femaleswhose follicles are in a different physiological state. Itis possible that if progesterone concentration wasincreased or decreased for females 2 and 7 (Table 1), ahigher percentage of ovulated oocytes would beobtained. Moreover, we are almost sure that 30 ng/mLof progesterone turned out to be an insufficient dosefor induction of ovulation in the hybrid between theRussian sturgeon and the Siberian sturgeon since avery high percent of ovulated oocytes was obtained inthe same experiment during treatment of follicles ofwith aciGTH (Table 4).

The possibility of a stable reproduction of ovulationof sturgeon oocytes in vitro is of interest not only forthe study of mechanisms of regulation of this processbut can be also important for the solution of importantapplied tasks. In the first place, we mean a possibilityto obtain offspring of rare and disappearing specieswithout a risk of loss immediately of all. This canoccur if a female is injected with a hormonal prepara�tion according to a scheme inadequate to its state.There is no reliable method for determination of thephysiological state of follicles that would guarantee theresult of the hormonal treatment (Goncharov et al.,1999). An alternative of the hormonal treatment of afemale is obtaining mature and ovulated eggs in vitro.Of course, it is important that matured and ovulatedovicells are capable of normal development.

After our study on stellate sturgeon (Goncharovet al., 2009), we got an impression that such simple saltmedium as RMS is not capable of promoting the for�mation or providing the maintenance of the capacityof eggs for development despite the fact that it is pos�sible to induce in it both maturation and ovulation ofoocytes. Recall that in the cited paper a considerably

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better result was obtained upon the use of the dilutedLeibovitz medium.

Our study demonstrated that, with a decrease in theconcentration of sodium bicarbonate in RMS andusing low doses of progesterone or aciGTH, it is pos�sible to obtain mature, ovulated, and capable of nor�mal development eggs. Note that this method has beenalready successfully applied by us to obtain matureovicells of Siberian sturgeon that subsequently wereused to obtain a gynogenetic offspring. The resultsturned out to be comparable with results on obtaininga gynogenetic offspring from eggs that matured underthe hormonal treatment in vivo (Grunina et al., 2010).

Subsequently, it seems necessary to define moreprecisely the conditions of cultivation of follicles andhormonal treatment providing the best result infemales with a different physiological state. It is quiteprobable that the best result can be obtained upon theuse of Leibovitz medium rather than RMS at a certainstate of follicles and the reverse at another state. At thepresent time, during work with unique females, itseems expedient to obtain in vitro mature, ovulated,and capable of development eggs to use in parallel alsothe described previously (Goncharov et al., 2009) anddescribed in this paper optimal conditions for cultiva�tion of follicles and their hormonal treatment.

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