1. Introduction
Assisted reproductive technologies (ART) aim to overcome human infertility and to optimize reproductive exploitation in domestic animals and the preservation in endangered species.
In a female context, most of ART’s success has been through the optimization of the use of fully-grown competent oocytes to generate high quality embryos to be transferred. This is contrasted by the fact that most individual reproductive resources are enclosed into primordial and growing oocytes. These female gamete reserves, constituting the largest proportion of the total follicular population in the ovary [
1,
2], cannot be enrolled in ART, even though it has been demonstrated that their biological potential is maintained in cryopreserved ovarian tissues of several mammals (in sheep, goats and humans [
3,
4,
5]).
One challenging biotechnological perspective is to mimic the process of follicle growth in vitro to support the acquisition of gamete meiotic and developmental competences, thus enlarging the availability on an individual basis of oocytes that can be enrolled with success in in vitro Maturation (IVM)/in vitro Fertilization (IVF) and embryo cultures. The
ivF approach, which is of great value in valorizing large sources of immature gametes, represents a breakthrough in the reproduction field for therapeutic, experimental, and diagnostic purposes. So far, the
ivF system has been developed successfully in mice [
6,
7] but it remains to be translated with efficiency to medium/large mammals. Currently, this limit is due to the longer period required for follicle/oocyte growth, the greater dimension of antral follicles enclosing competent oocytes and the difficulty in mimicking the environmentally favorable conditions that guarantee a synergic oocyte and somatic compartment development by preserving the tissue architecture [
8,
9,
10,
11,
12,
13,
14,
15,
16,
17]. To translate the in vitro culture systems to non-rodent models, a better comprehension of the physiology mechanisms triggering follicle growth initiation, sustaining the interplay between germinal and somatic compartment, and supporting the basic metabolic needs of early growing follicles, are required.
For this reason, even if primordial follicles represent the ideal starting source of gametes for
ivF [
1,
2], most attention has been focused on medium/large mammals for the validation of the protocols supporting the growth of preantral (PA) follicles in order to reduce the cultural period required to complete the phase of oocyte growth [
3,
8,
9,
12,
13,
14,
18,
19].
The in vitro growth of isolated PA follicles [
17,
20,
21,
22,
23], as well as the in vitro PA growth of follicles enclosed in strips of ovarian cortex [
16,
24,
25], particular by culturing isolated PA follicles under two-dimensional (2D) and three-dimensional (3D) systems, has been successfully developed in non-rodent mammals and humans by several research groups.
Notably, the small ruminant model plays a pivotal role in this research context [
12,
24,
26,
27,
28,
29,
30,
31]. This is mainly due to its high translational value. Indeed, profound similarities with human reproductive physiology have been recognized through the small ruminant model: they are medium-sized mammals, mainly mono-ovulatory species; and the female gamete achieves evolutionistic growth during its transition from PA to early antral (EA) follicle [
32,
33,
34]. In addition, researchers enjoy access to a substantial number of biological samples using the small ruminant model since the ovaries of small ruminants, even prepubertal animals, can be easily collected from slaughterhouses as discarded tissues. The high translational value of the medium-sized ruminant model is emphasized even further when using prepubertal ovaries, which exactly mimic the physiological structure of girls and young women [
17,
35].
Studies that have focused on the in vitro development of PA follicles in small ruminants have demonstrated the possibility of stimulating follicle growth in culture by inducing the activation of steroidogenesis and the secretion of follicle bioactive factors (Inhibin: sheep [
23], goat [
26,
27]; or anti-Müllerian hormone [
31]). However, several pieces of evidence, obtained by comparing the functional status of in vivo and in vitro developed follicles, showed that the current available protocols induced an earlier differentiation status, causing an increase in the rate of granulosa cell proliferation [
31], the acceleration of follicular cell specialization [
17], an earlier and faster acquisition of follicular maturation markers (
CYP19A1,
FSHR,
ESR2,
INHA,
INHBA,
INHBB, and
FST), and the upregulation of theca-related genes (
LHR,
CYP17A1, and
CYP11A1) [
31].
The acceleration induced in follicle by the in vitro conditions may be responsible for the impaired follicles–oocyte dialogue affecting the in vitro oocyte growth process (buffalo [
8], sheep [
17], and goat [
12]) and probably a precocious reduction in cumulus-oocyte metabolic coupling [
17,
31].
Cadoret et al. [
31] demonstrated in sheep that the expression of oocyte-specific genes
ZP3,
GJA4 (encoding the connexin 37 protein),
KIT, and
BMP15 were significantly downregulated during culture.
However, despite the difference recorded between follicle growth in vivo and in vitro, enclosed oocytes may express growth capacity and a progressive degree of epigenetic maturation [
17] associated with the acquisition of meiotic competence [
3,
17]. Sheep oocytes grown in vitro, indeed, were able to resume and complete meiosis, leading to meiotic competence that was equivalent to [
17], and sometimes higher than, that of oocytes derived from early antral follicles developed in vivo [
31,
36].
In addition, despite the alterations recorded in oocyte gene expression [
31], developmental competence has also been demonstrated in several ruminant models, such as bovine [
37], buffalo [
8], sheep [
17], and goat [
12] models.
The factors driving the PA to antral follicle transition are still poorly understood, but in most of in vitro systems, Follicle Stimulating Hormone (FSH) stimulation appeared to be crucial in inducing coordinated follicle and oocyte development. Recent studies used the responsiveness of the PA follicle gonadotropin alone or in combination with other growth factors [
38,
39,
40,
41,
42]. Conversely, FSH in vivo seems to be a dispensable factor in follicles reaching the preantral antral stage and in the proliferation of granulosa cells, [
2,
43] and is controlled by intraovarian regulators [
44], while follicles acquire FSH dependence during the transition from the preantral to the antral stage [
45,
46], where gonadotropin plays a pivotal role in regulating follicular fate [
44]. Accordingly, a heavy requirement of FSH dependence was also reported in vitro in the stimulation growth from the PA stage to the antral stage [
38,
47].
In particular, FSH in small ruminants was recognized to induce in vitro PA follicle growth, antrum differentiation, steroidogenesis activation [
17,
31,
48], the release of survival factors protecting the granulosa from apoptosis, and to stimulate the enhancement of gap junctional communication [
4,
23,
49,
50,
51]. In addition, several pieces of evidence demonstrated that the in vitro influence of FSH on PA follicle growth is strictly dose-dependent.
While a low gonadotropin concentration seems to be required to promote oocyte growth [
52], high concentrations of FSH enhance follicle development but have deleterious effects on the developmental capacity of oocytes [
17,
52]. Moreover, other factors seem to influence follicular performance, such as the use of sequential FSH concentration [
30] and/or the different FSH origin [
53].
However, besides the biological evidence obtained by different research groups regarding the effects of FSH, the debate continues.
For veterinary purposes, in order to induce the growth of follicles by ovaries and to increase the ovulation rate before the artificial insemination, Equine Chorionic Gonadotropin (eCG), previously named Pregnant Mare Serum Gonadotropin (PMSG), has been used successfully. Equine Chorionic Gonadotropin is a placental glycoprotein obtained from the serum of pregnant mares. It is widely used as a valid substitute for species-specific FSH in female mammals due to its longer circulatory half-life, its FSH like-activity, its easier use as a trans-species hormone, and its limited costs [
54,
55].
According to this premise, the research reported in this paper aimed to study the influence of two different gonadotropins (FSH vs. eCG) on in vitro follicle cultures of a single ovine PA follicle, collected at the large secondary stage. The ivF outcomes obtained by using the species-specific ovine FSH (oFSH) or the trans-species chorionic gonadotropin (eCG) were analyzed after 14 days of culture by comparing the FBS-supplemented medium with the FBS-free medium, follicle/oocyte in vivo/in vitro growth, the timing and percentage (%) of antrum differentiation, the results of an estradiol production assay, gene expression, as well as the percentage of Metaphase II (MII) oocytes after IVM.
3. Discussion
Over the past decades, follicle culture systems have been developed for promoting ovarian ivF in several mammalian species to reproduce in culture the growth of meiotic incompetent oocytes. Despite the high in vitro maturation rate achieved in current
ivF protocols, the effect of some aspects, such as those related to epigenetic and environmental stimuli, on the oocyte quality need to be further investigated [
59]. However, this advanced reproductive technique represents a potential strategy to improve animal reproduction/production and research trials, as well as to preserve human fertility. The biomedical interest in
ivF has been further increased recently as a consequence of the increased rate of survival of young patients affected by cancer [
60]. In this case, setting up validated
ivF protocols represents a safe biotechnical strategy through which to restore fertility chances once patients reach adult life in a healthy condition instead of the transplantation of their ovarian tissues, which potentially exposes the organism to the risk of reintroducing malignant cells [
61,
62].
However, the ability to reproduce in vitro the first phases of folliculogenesis (follicle growth and oocyte development) still represents an unsolved challenge for reproductive biotechnology in mammals [
17,
23,
63,
64] as it requires wide margins of standardization, including the definition of the gonadotropic role [
60].
FSH seems to exert an essential influence in vitro also during the phase preceding antrum cavity formation, when this hormonal stimulation has been considered essential to establish coordinated growth between the germinal and somatic compartments [
47]. At the same time, FSH constrains
ivF implementation due its high species specificity and its variable biological activity, which related to its source (recombinant or naïve). To overcome this limit, it might be of interest to study more handling solutions to promote
ivF by substituting its stimulatory influence [
65] or, alternatively, by adopting, as for animal superovulation, a chorionic gonadotropin with FSH-like activity.
A placental glycoprotein hormone, eCG is secreted by fetal trophoblastic epithelial cells constituting the endometrial cups [
66]. It demonstrates biological activity similar to FSH, physiologically causing follicles ovulation and leading to produce accessory corpora lutea which helps to support the developing fetus [
67].
For a long time, eCG was used as an alternative FSH hormone for superovulation induction by improving the process of follicle recruitment in several mammal models [
68]. Indeed, it was successfully used in small mammals, such as mice rats [
69] rats alone [
70], medium-large sized mammals such as sheep [
71,
72,
73], goats [
74], cows [
75], mares [
76], and pigs [
77].
Notably, its use has been consolidated over recent years due to its longer circulatory half-life, enhanced trans-species effect, lower cost, and greater commercial availability compared with FSH [
54,
55]. Despite these numerous advantages, no evidence has been collected to date documenting eCG’s influence on the promotion of in vitro folliculogenesis.
Based on our results, 4 IU/mL eCG was found to efficiently support proper ovine PA follicle growth by inducing the synchronous development of germinal and somatic compartments. Furthermore, eCG appears much more effective in improving some key cultural biological targets of ivF, such as follicle growth, estradiol synthesis, and the acquisition of oocyte meiotic competence. Overall, the evidence presented in this paper suggests widespread eCG use as a substitute for oFSH in ivF protocols.
In more detail, while eCG and FSH demonstrated a similar biological influences on the percentage of antrum formation, healthy follicles, and recovered oocytes, conversely, chorionic gonadotropin supplementation showed the ability to significantly enhance the incidence of follicles with greater rates of growth (Δ growth). Indeed, almost all the eCG-treated follicles (81%) displayed a Δ growth greater than that of the average PA follicles’ Δ growth (>40%). The achievement of such of growth rates positively influenced the oocytes’ quality.
Indeed, oocytes expressing the highest IVM meiotic competence (68% MII in eCG vs. 43% MII with oFSH) were isolated from precisely this category of EA follicles (PA follicles with a rate of growth > of medium Δ growth). This effect was observed either when
ivF was carried out in the FBS-supplemented medium or under improved
ivF conditions, such as those performed using the FBS-free medium on. Notably, by coupling the use of eCG with the beneficial effect of FBS-free medium supplementation, the performance of meiotic competence acquisition was similar to that of the oocytes collected from the in vivo EA follicles (75.5% vs. 77.7% MII oocytes, respectively). Accordingly, Park et al. demonstrated how the replacement of FBS-supplemented medium with an FBS-free medium exerted a useful effect on oocyte maturation systems, contributing to identifying the functional elements necessary for folliculogenesis [
58]. Notably, the use of FBS-free medium supported higher follicular survival rates when compared to the medium supplemented with FBS [
56].
In our study, the FBS-free medium was able to positively affect follicle development by equalizing the timing of antrum formation and shortening the window period needed for the antrum cavity development regardless of whether the treatment was eCG or oFSH. In addition, all the follicles that developed an antrum cavity in FBS-free medium showed a Δ growth of more than 40% (for eCG: 76% and for oFSH: 66%).
This effect may have been the result of a longer in vitro persistence or a greater degree of metabolic coupling between the somatic and germinal compartments under reduced concentrations of FBS [
17,
64]. Any information on oocyte–follicle coupling were collected in the present research; however, indirect evidence may confirm this pro-healthy effect of FBS removal. Indeed, focusing attention on the in vitro grown follicles developing antrum cavity, those treated with eCG in combination with FBS free-medium, showed a marked expression of GJA1. The greater expression of GJA1 may be indicative of better support in the synchronization of the process involving the proliferation of granulosa cells, indirectly suggesting that treatment with eCG guarantees an efficient coupling of the somatic and germinal compartments, which in turn reflects the observed meiotic competence acquisition performance. A lower expression in those follicles that did not develop an antrum cavity during e
ivF could be justified as a lack of communication between oocyte and granulosa cells. Indeed, the PA follicles collected after 14 days of culture in the FBS-supplemented medium showed a higher incidence of degenerated follicles (approximately 40% in
ivF FBS-supplemented medium vs. 34% and 24% in follicles treated with oFSH and eCG in FBS-free medium) as well as a smaller percentage of EA (for oFSH and eCG respectively: 58% and 61% in FBS-supplemented medium vs. 66% and 76% in the FBS-free medium). Moreover, it was possible to notice that the antrum cavity differentiation recorded in the follicles treated with eCG and cultured in the FBS-free medium led to the in vitro achievement of a high degree of follicular maturity, as evidenced by the steroidogenic gene expression profiles and the estradiol release in the culture medium.
More specifically, the superior in vitro performance of eCG in sustaining the follicular wellness in combination with the FBS-free medium was confirmed by the activation of the transcriptional steroidogenic program and by the secretion of E2. Notably, the steroidogenic enzymes CYP17A1 and CYP19A1 were upregulated in those follicles developing antrum cavity in both treatments. However, eCG was able to activate the steroidogenic transcriptional program with higher efficiency, as demonstrated by the more evident and significant expression of steroidogenic enzymes in the follicles that developed antrum cavity compared to that recorded in the oFSH.
The superior performance of eCG may have been due to its LH-like action, as demonstrated by the recorded upregulation of the theca-specific CYP17A1 gene. Moreover, our data clearly suggest that antrum development represents a key factor in the modulation of CYP17A1 expression, since only the follicles with an antrum cavity showed a significant upregulation of this gene. Consistently, CYP19A1 upregulation could be interpreted as a result of a boosted production of precursors for estrogen synthesis as well as a crosstalk between oocyte, granulosa, and theca cells, representing a key element of follicular well-being. Importantly, E2 secretion in the conditioned culture media showed a coherent trend of accumulation. The greater functional maturation of the follicles that developed an antrum cavity in the presence of eCG in the FBS-free medium also positively reflected improved oocyte development, especially in terms of the acquisition of meiotic competence.
In light of this evidence and previous studies that p documented a correlation between antrum cavity formation and follicular maturation degree [
17,
31,
64,
78,
79,
80] the use of antrum cavity formation as a biological marker of successful follicular growth could be hypothesized.
Moreover, the synergistic FSH and LH-like activity of eCG, was documented to represent a key element of proper follicle growth, ovulation, and corpus luteum formation [
81,
82].
The stimulatory effect of eCG was validated by verifying the ability of chorionic gonadotropin to improve the outcomes of the in vitro follicular development, increasing the in vitro follicle growth rate % and enhancing the gene expression of steroidogenic enzymes and estradiol releasing. Similarly, eCG promoted synchronous oocyte growth and follicle development, as demonstrated by its ability to induce an efficient meiotic resumption, reaching IVM outcomes similar to those recorded in the in vitro maturated oocytes derived from the in vivo grown EA follicles. Many other advantages resulting from the use of eCG to support in vitro follicle culture should also be considered: its commercial availability, longer circulatory half-life, trans-species action, and lower costs. Moreover, chorionic gonadotropin can extend its biological power to in vitro cultural systems according to its FSH-like activity, as successfully demonstrated in validated superovulation protocols (
Figure 9).