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Theriogenology 74 (2010) 557–562 www.theriojournal.com
Newborn pig ovarian tissue xenografted into Severe Combined Immunodeficient (SCID) mice acquires limited responsiveness to gonadotropins L.F. Pisania,d, G. Pennarossaa, E. Papasso Brambillaa, M.M. Rahmana, G. Lazzarib, A. Zecconic, T.A.L. Brevinia, F. Gandolfia,* a
Department of Animal Science, Laboratory of Biomedical Embryology, Università degli Studi di Milano, 20133 Milano (Italy) b AVANTEA srl, Laboratorio di Tecnologie della Riproduzione 26100 Cremona, Italy c Department of Animal Pathology, Hygiene and Veterinary Public Health, Università degli Studi di Milano, 20133 Milano (Italy) d Current address: Department of Animal Pathology, Hygiene and Veterinary Public Health, Università degli Studi di Milano, 20133 Milano (Italy) Received 6 October 2009; received in revised form 17 February 2010; accepted 22 March 2010
Abstract In the pig ovary, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles can be observed, for the first time, at ⬃60 –90 d of age. At approximately the same time, secondary follicles become responsive to gonadotropins, leading to the formation of antral follicles. Placing pieces of ovarian tissue under the kidney capsule of immunodeficient (SCID) mice allows the requirements for follicular recruitment and development to be studied. The objective of this study was to investigate if primordial follicles contained in ovarian fragments isolated from newborn piglets (36 ⫾ 12 h old) and immediately transplanted under the kidney capsule of SCID mice, are able to become responsive to gonadotropins after 60 d (as in an unaltered animal). Ovarian fragments were transplanted under the kidney capsule of three groups of four female and four male SCID mice. The first group did not receive any hormonal treatment for 12 wk. The second group was treated from the 9th week with 1 IU of FSH/LH on alternating days for 3 wk, and the third group was treated with 5 IU Pregnant Mare Serum Ganadotropin (PMSG) 48 h before euthanasia. Primordial follicles contained in ovarian fragments isolated from newborn piglets developed only to the secondary stage. Therefore, development of gonadotropin responsiveness in ovarian fragments xenotransplanted in SCID mice was delayed compared to what occurs in the unaltered animal, and there was minimal response to exogenous gonadotropins. © 2010 Elsevier Inc. All rights reserved. Keywords: Follicle development; Ovarian transplant; Ovary; SCID mice; Xenografting
1. Introduction The ovarian follicle reserve is gradually depleted during reproductive life; typically, only a minute fraction of the initial population reaches full development * Corresponding author. Tel.: ⫹390250317990. fax: ⫹390250317980. E-mail address:
[email protected] (F. Gandolfi). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.03.017
and ovulation. The recruitment of primordial follicles is crucial for the process of folliculogenesis, both in vivo and in vitro. Whereas the later stages of folliculogenesis are well characterized, little is known regarding mechanisms that regulate the onset of follicular development from the primordial follicle stage. The transition from primordial to primary follicle occurs independently of a direct action of gonadotropins [1] and it
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commits a follicle to development or atresia [2]. The mechanisms driving this transition remain largely unclear, because it is impossible to study in vivo, and attempts to replicate it in vitro have been largely unsuccessful. Ovarian grafting provides an alternative method for the maturation of oocytes in primordial follicles of large mammals. Ovarian tissues have been prepared from a wide range of species, including humans [3– 6], dogs [7], monkeys [8], sheep [9], cows [10], pigs [11– 13], tammar wallaby [14], and common wombats [15,16] and xenografted to immunodeficient mice. In pigs, the transition from primordial to primary and secondary ovarian follicles begins before birth, but antral follicles were first detected at ⬃60 d of age [17]. At approximately the same time, 9 wk of age, secondary follicles become responsive to exogenous gonadotropins [17,18] and there was a constant decrease in egg nests, which constituted the earliest recognisable form of gamete cells in the ovary [17,19]. The transplantation of pig ovarian fragments into SCID mice showed that primordial follicles of this species can develop and become responsive to exogenous gonadotropins, with follicles growing as large as 2 mm in diameter. Moreover, the oocytes contained in these follicles acquired the ability to resume meiosis, be fertilized, and cleave, up to the blastocyst stage [11, 13,20]. In these experiments, tissue was collected from 20 d old piglets. There are no data regarding whether primordial follicles collected from pigs immediately after birth become gonadotropin-responsive after transplantion into SCID mice. The objective of the present study was to determine if primordial follicles contained in ovarian fragments isolated from newborn piglets and immediately transplanted under the kidney capsule of SCID mice were responsive to gonadotropins after 60 d (the onset of responsiveness in intact animals). Furthermore, we determined the effects of gender of the recipient mouse, as well as two gonadotropin treatments for stimulation of xenografted ovarian fragments. 2. Materials and methods 2.1. Collection of ovarian tissues Both ovaries were collected from three newborn Large White piglets (36 ⫾ 12 h old) in saline solution. The cortical region of each ovary was dissected with surgical blades into 1 mm3 fragments. Nine fragments from each ovary of each piglet were randomly selected.
One fragment was fixed immediately, whereas the others were pooled and used for xenografting. 2.2. Xenografting As recipients, 12 male and 12 female Severe Combined Immunodeficient Mice (SCID), 6 – 8 wk old (Charles River Laboratories, Calco Italy), were used. Before xenografting, mice were anesthetized (22 mg/kg of tribromoethanol given IP; Sigma Aldrich, Milan, Italy) and each kidney was exteriorized through a dorsal-horizontal incision. One fragment of ovarian tissue was randomly selected from the pool and inserted under each kidney capsule (two fragments for each mouse). Mice were housed in filter-topped cages in a positive pressure room (temperature, 22–24°C), 12/12 h darklight, with free access to clean water, and balanced feed pellets. The duration of xenografting was 12 wk. 2.3. Hormonal treatments Mice were randomly allocated into three groups, each composed of four males and four females, and each group receiving a treatment previously reported to support the formation of preantral and antral follicles. The first group, used as a control, did not receive any hormonal treatment for 12 wk [12,21]. The second group was treated from the 9th week with 1 IU FSH/LH (Pluset, Serono, Rome, Italy) on alternating days for 3 wk (modified from [3,20]). The third group was given 5 IU Pregnant Mare Serum Gonadotropin (PMSG, SigmaAldrich, Italy) 48 h before euthanasia [20,22]. 2.4. Histological analysis Ovarian fragments collected from the newborn piglets immediately after death, and the region of each mouse kidney containing the ovarian fragment, isolated from the rest of the organ after death, were fixed in 4% paraformaldehyde, and embedded in paraffin wax. Serial sections (5 m thick) were taken from each sample at 100 m intervals. Sections were stained with hematoxylin-eosin, and follicular structures were evaluated for morphological normality and stage of development. Follicles were scored, in a blind fashion by a single operator, on each set of serial sections, as previously described [23], according to criteria adapted from Paynter et al. [24]. These were as follows: primordial follicles (germinal vesicle-stage oocyte surrounded by a single layer of flat cells); primary follicles (small follicles containing an oocyte surrounded by one or two layers of cuboidal granulosa cells); secondary follicles (larger structures, with several layers of granulosa cells surrounding a germinal vesicle-stage oocyte); and an-
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Table 1 Total number of ovarian follicles in each developmental stage and growth rates in following transfer of neonatal porcine ovarian tissue to male and female SCID mice subjected to various hormonal treatments. Treatment
Control FSH/LH PMSG
Male
Female
Primordial (%)
Primary (%)
Secondary (%)
Growth rates* (%)
245 (73.4)a,1 472 (69.7)a,1 328 (73.9)a,1
54 (16.2)a,1 119 (17.6)a,1 60 (13.5)a,1
35 (10.5)a,1 86 (12.7)a,1 56 (12.6)a,1
26.6 30.3 26.1
Primordial (%)
Primary (%)
Secondary (%)
Growth rates* (%)
173 (81.6)a,2 185 (76.8%)a,2 222 (75.3)a,1
33 (15.6)a,2 35 (14.5)a,2 69 (23.4)b,2
6 (2.8)a,2 21 (8.7)b,2 4 (1.4)a,2
18.4 23.2 24.7
* Proportion of primordial follicles that were recruited to become primary and secondary follicles. Within a column, means without a common superscript differed (P ⬍ 0.05). 1,2 Within a specific endpoint, the lack of a common superscript indicates a difference between male and female mice (P ⬍ 0.05). a,b
tral follicles (with an antrum of variable size visible as a single cavity). Five sections of each fragment collected from each ovary of the three newborn piglets (total of six fragments and 30 sections) were analysed to determine the characteristics of the follicle population before xenotransplantation. Similarly, all follicles found in five sections of each fragment xenotransplanted under the kidney capsule of each individual (four males and four females) of each treatment group (control, FSH/LH, and PMSG) were scored (total of 48 fragments and 240 sections). Follicles were counted and proportions of each follicle type (based on the total number of follicles for each piglet or each treatment group) were calculated. Results were expressed as proportions and ranges of follicles in each developmental stage, as previously described [23,24]. 2.5. Data analyses Statistical differences among follicle proportions were assessed by 2 test with Yates’ correction, using StatXact 6.0 software (Cytel Corp, Cambridge, MA, USA) considering, for each experimental group, either male and female categories separately, or all mice combined. The statistical analysis was performed applying exact tests and, therefore, the results were reliable, even when cell frequencies were low. Follicle distribution among piglets (at the time of collection), was assessed by a multinomial distribution goodness of fit test. 3. Results
Fig. 1. Representative pictures of newborn piglets ovarian fragments before (A) and after (B) 12 wk under the kidney capsule of a SCID mouse. 1 ⫽ primordial follicle; 2 ⫽ primary follicle; 3 ⫽ secondary follicle.
All data are summarized in Table 1. 3.1. Fresh ovarian tissue analysis A total of 564 follicles were present in the examined sections (Fig. 1A). Primordial follicles accounted for
the vast majority of the total population (95.21%, range 93.62–96.43) with a small percentage of primary follicles (3.90%, range 2.04 –5.85) and the presence of a few secondary follicles (0.53%, range 0.51– 0.54) and
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germ cell nests (0.36%, range 0 –1.2). There were no significant differences among individuals. 3.2. Xenografting analysis Ovarian xenografts were examined 12 wk after transplantation; they were distinguishable and recovered in 24 of 24 mice. Based on histological examination of the fragments, none exhibited structural damage, and the ovarian stroma was devoid of necrotic cells. No signs of immune reaction were visible (Fig. 1B). 3.3. Follicle development In all experimental groups, irrespective of the hormonal treatment or gender of the recipient mouse, follicle development reached the secondary stage, but no antral follicles were observed. At the end of the xenotransplantation period, the proportion of primordial follicles that were activated to the primary and secondary stage, in each treatment group, ranged from 18.4 to 30.3%, an increase (P ⬎ 0.01) from the 4.4% (range 2.5– 6.4) present in ovarian fragments at the time of the xenografting procedure. No egg nests were detected at this stage. The gender of the recipient SCID mouse and the hormonal treatment influenced the response of pig ovarian fragments in different ways. In the absence of hormonal treatment and after treatment with FSH/LH, the proportion of primordial follicles was lower in male than in female mice, whereas there was no significant difference between the two genders after PMSG treatment. Furthermore, the lower rate of primordial follicles in FSH/LH stimulated male mice was sufficient to make this treatment significantly different combined for both genders. The higher proportion of primordial follicles in female mice with no hormonal treatment and after treatment with FSH/LH was reflected in a higher rate of primary follicles in male mice. On the contrary, ovarian fragments recovered from PMSG-treated females contained a higher rate of primary follicles than their male counterparts. This, in turn, was manifested in a lower rate of secondary follicles in female versus male PMSG-treated mice. Furthermore, there was a higher proportion of secondary follicles in male mice in the absence of hormonal treatment, whereas the high proportion in the FSH/LH-treated male group was not significantly different from the female group, making this the group with the greatest overall development of secondary follicles.
4. Discussion We investigated whether newborn pig ovarian tissue, xenotransplanted into a SCID mouse, can develop responsiveness to gonadotropins in the same timeframe required in vivo. An important factor for the successful transplantation of ovarian cortex in immunodeficient mice is the quick establishment of rich blood supply. Revascularization of the graft is crucial for the survival of ovarian follicles after xenografting. The recovery of transplanted ovarian tissue from all recipient mice and the absence of any rejection or necrotic lesion confirmed that the profuse blood supply in the subcapsular region of the kidney makes this an excellent site for transplantation [10,11]. Pig primordial ovarian follicles are observed for the first time in the deepest part of the cortex, next to the medulla around 56 d post coitum (dpc) [25,26] and at this stage of development, follicle somatic cells are not endocrinologically active, nor do they express FSH receptors until follicle growth is initiated [27]. The situation remains basically unchanged until ⬃60 d after birth, when the development of multilayer secondary follicles is observed for the first time in gilt ovaries [28]. A pig primordial follicle requires ⬃84 d to reach the antral stage [29]. Similarly, ovarian fragments collected from 20 d old piglets and transplanted into ovariectomized female SCID mice for ⬃60 d formed antral follicles, even in the absence of gonadotropins, albeit at a low rate [11]. On the contrary, based on the present study, primordial and primary follicles in ovarian fragments from newborn piglets were unable to reach the antral stage when xenotransplanted into a SCID mouse for 84 d (the interval required to reach this stage in the unaltered animal [29]). Large secondary follicles are able to synthesise FSH receptors, so they can respond to the natural changes in serum gonadotropin concentrations, as well as to exogenous hormonal treatment [30,31]. We initiated treatment with FSH/LH at the same time (63 d) but the lack of development of antral follicles indicated that responsiveness to endogenous and exogenous gonadotropins was delayed in xenotransplanted ovarian fragments. Furthermore, treatment with PMSG 3 wk later gave the same result, with no follicles developing beyond the secondary stage. Primordial follicles present in ovarian fragments isolated from 6 mo old pre-pubertal gilts were unable to form antral follicles when kept for 2 mo under the kidney capsule of SCID mice, whereas some antral follicles were present in ovarian tissue obtained from 20 d old piglets xenotransplanted in the same
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conditions [13]. Perhaps there is a time window during which primordial follicles remain responsive to gonadotropins or other stimuli upon xenotransplantation into a SCID mouse. Overall, hormonal treatment had very limited effects on follicular development in the present study. In that regard, follicles reached only the secondary stage, when their development is still largely independent of gonadotropin stimulation. Therefore, we inferred that the vast majority of the follicles in the xenotransplants were unable to form functional ganadotropin receptors. Since only a small piece of ovarian cortex was used for transplantation, perhaps there was a relative deficiency of stroma and/or of endogenous androgen production as substrate for estrogens. The fact that follicular development observed in fragments retrieved from males was overall better than in female recipients gave credence to this hypothesis. Similarly, Weissman et al [4] demonstrated that male mice supported follicle development in human ovarian fragments at higher rates than females. In summary, we established that primordial follicles contained in ovarian fragments, isolated from newborn piglets, develop to the secondary stage after being xenotransplanted into SCID mice for 12 wk. Therefore, development of gonadotropin responsiveness was delayed, compared to intact animals. Consequently, no major effects of administering exogenous gonadotropins to the host mice were observed. Further investigations are required to determine what changes are necessary in order to acquire this responsiveness. Acknowledgements The first two authors contributed equally to this work. References [1] Meduri G, Charnaux N, Driancourt MA, Combettes L, Granet P, Vannier B, Loosfelt H, Milgrom E. Follicle-stimulating hormone receptors in oocytes? J Clin Endocrinol Metab 2002;87: 2266 –76. [2] van den Hurk R, Zhao J. Formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles. Theriogenology 2005;63:1717–51. [3] Oktay K, Newton H, Mullan J, Gosden RG. Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulating hormone. Hum Reprod 1998;13:1133– 8. [4] Weissman A, Gotlieb L, Colgan T, Jurisicova A, Greenblatt EM, Casper RF. Preliminary experience with subcutaneous human ovarian cortex transplantation in the NOD-SCID mouse. Biol Reprod 1999;60:1462–7.
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