Microvascularization of Corpus Luteum of Bovine Treated
With Equine Chorionic Gonadotropin
CARLOS EDUARDO BEZERRA MOURA,1* NATHIA NATHALY RIGOGLIO,2JANINE KARLA FRANC¸A S. BRAZ,3 MARCELLO MACHADO,4PIETRO S. BARUSELLI,5ANDPAULA DE CARVALHO PAPA2
1Department of Animal Sciences, Federal Rural University of Semiarid Region (UFERSA), Mossoro, RN 59625-900, Brazil 2Department of Surgery, School of Veterinary Medicine and Animal Science, University of S~ao Paulo, S~ao Paulo, SP 055082270,
Brazil
3Department of Morphology, Bioscience Center (BC), Federal University of Rio Grande Do Norte (UFRN), Natal, RN 59078-970, Brazil 4Department of Biological and Veterinary Sciences, Campus Curitibanos, University of Santa Catarina, Curitibanos SC 89529-000,
Brazil
5Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of S~ao Paulo, S~ao Paulo,
S~ao Paulo, Brazil
KEY WORDS bovine; corpus luteum; equine chorionic gonadotropin; microvascular density ABSTRACT This study aimed to evaluate the morphological changes in microvascular den-sity and corpus luteum (CL) vascularization in cows treated with eCG during stimulatory and superovulatory protocols. Sixteen cows were synchronized and divided into three groups: control (n 5 6), stimulated (n 5 4) and superovulated (n =6), one was submitted to estrous synchroniza-tion (ES) and received no eCG (control), and those that were submitted to ES and received eCG before or after follicular deviation (superovulation and stimulation of the dominant follicle, respectively). Ovulation was synchronized using a progesterone device-based protocol. After six days of ovulation, the cows were slaughtered and the ovaries and CL were collected. The CLs were processed and photomicrographs were taken under light microscopy to assess the vascular volume density (Vv) by stereology, and scanning electron microscopy (SEM) was used to perform ultrastructural analysis of the microvasculature. The Vv in stimulated and superovulated cows significantly increased (P 0.0001) when compared to control, indicating that the eCG is able to induce angiogenic activity in bovine CL. However, no significant differences were observed between stimulated and superovulated cows. The SEM demonstrated ratings indicative of angio-genesis, marked by several button-shaped projections in the capillaries, and the presence of more dilated capillaries in CL treated with eCG. These morphological findings are evidence of an angiogenic effect of the eCG treatment in CL of cows. Microsc. Res. Tech. 78:747–753, 2015. VC2015 Wiley Periodicals, Inc.
INTRODUCTION
The corpus luteum (CL) is an endocrine gland that participates in the regulation of the ovarian cycle and fertility of the female (Schams and Berisha, 2004). Although lasting only 18 days during the bovine estrous cycle, the CL undergoes significant structural changes in this period, such as rapid proliferation and differentiation of steroidogenic cells and extensive angiogenesis (Skarzynski et al., 2013).
There is substantial evidence that the pituitary gonadotropins, FSH and LH, influence the expression of angiogenic agents in CL (Fraser and Wulff, 2003; Papa et al., 2007; Fatima et al., 2013a). LH stimulates the production of most of these factors by luteal cells in a paracrine mechanism that induces angiogenesis in the developing CL (Fraser and Wulff, 2003). FSH also increases the expression of VEGF in human luteal cells (Reisinger et al., 2007), and has caused a signifi-cant increase in the vascular density of buffalo CL (Papa et al., 2007). Other studies have also demon-strated that human chorionic gonadotropin (hCG) also promotes an increase in the expression of factors such
as VEGF and Ang in luteal cells (Christenson and Stouffer, 1997; Laitinen et al. 1997; Wulff et al., 2001).
The equine chorionic gonadotropin (eCG) is a gona-dotropic hormone produced by trophoblast cells of endometrial cups in pregnant mares and plays a major role in the maintenance of early gestation (Legardinier et al., 2005). In non-equine species, the eCG has both FSH and LH activity and stimulates follicular develop-ment, ovulation, and CL formation. This hormone has also been widely used together with progesterone ther-apy and pituitary stimulation by GnRH to shorten the postpartum anestrus in cows (Lunenfeld, 2004). There are reports that the eCG is capable of inducing growth
*Correspondence to: Carlos Eduardo Bezerra Moura, Departamento de Ci^ en-cias Animais, UFERSA, Av. Francisco Mota, 572. Bairro Costa e Silva, 59625-900, Mossoro, RN, Brazil. E-mail: carlos.moura@ufersa.edu.br
Received 22 February 2015; accepted in revised form 24 May 2015 REVIEW EDITOR: George Perry
Contract grant sponsor: FAPESP; Contract grant number: 2008/58837-5; Con-tract grant sponsor: CAPES.
DOI 10.1002/jemt.22533
and ovulation of follicles in pre-pubertal heifers (Duffy et al., 2004; Ferreira et al., 2013).
Recently, an increase in CL volume and plasma pro-gesterone concentration during the subsequent luteal phase in cows treated with eCG has been reported (Ferreira et al., 2013; Rostami et al., 2011), and this fact has been related to the influence of this hormone on the differentiation and function of granulosa cells and theca after ovulation (Kenyon et al., 2012), as well as on the number of large and small luteal cells, and the volume and shape of mitochondria in these steroi-dogenic cells (Rigoglio et al., 2013). However, until now no studies have evaluated the microvasculature of the CL from cows treated with eCG, despite its blood sup-ply being closely related to its steroidogenic function. Therefore, this study aimed to evaluate the changes caused by stimulatory and superovulatory treatments with eCG in microvascular density and vascularization of bovine CL.
MATERIAL AND METHODS Animals
We studied 16 crossbred cows (Angus x Nelore) aged between 2 and 5 years which were kept in pasture and supplemented with ground corn (17.03%), soybean meal (4.65%), urea (1.13%), ammonium sulfate (0.11%), minerals (1.69%), salt (0.53%) and corn silage (fodder) (74.86%). These animals were evaluated for body condition score (1-5 scale, according to Wildman, 1982 and ovarian status by rectal palpation, as described by Madureira, 2004), and those with scores between 2.0 and 3.0 and with normal ovarian activity (CL presence and/or absence of follicles and cysts) were selected according to Fatima et al., (2013b).
All experimental procedures performed in this work were approved by the Ethics Committee for Animal Use of the School of Veterinary Medicine and Animal Science, University of S~ao Paulo (protocol n 8 1637/ 2009).
Hormonal Treatment
For hormone treatment, females were randomly divided into three groups: control (n 5 6), stimulated (n 5 4) and superovulated (n 5 6). This treatment con-sisted of the use of a vaginal implant containing 1g of progesterone (Bovine Intravaginal Device; progester-one: 1 g; Primer, Technopec, S~ao Paulo, SP, Brazil) and 2 mg of IM estradiol benzoate (Estrogen Farmavet, S~ao Paulo, SP, Brazil) administered to all animals on Day 0 of the protocol. For control and stimulated cows, the implants were removed and 0.15 mg ofD
-cloproste-nol (PGF2a; Prolise; Arsa, Buenos Aires, Argentina) was administered on Day 8. In stimulated cows, 400 IU of eCG (Novormon; Syntex, Buenos Aires, Argen-tina) was also administered on Day 8, which is consid-ered to be the optimal dose to improve dominant follicles (SaFilho et al., 2010). The control cows did not receive any eCG. At 48 h after removal of the implants, control and stimulated animals received 0.025 mg lec-irelin (GnRH; Gestran Plus, Arsa). Superovulated cows were treated with 2000 IU eCG on day 4 and 0.15 mg ofD-cloprostenol on Day 6. In this group, the
implants were removed on day 7, then a second dose of 0.15 mgD-cloprostenol was administered at this time
and after 12 h they received 0.025 mg of GnRH
(Baru-selli et al. 2011). According to Baru(Baru-selli et al., (2012), differences regarding hormonal protocol for superovu-lated cows are based on achieving more synchronous ovulations. Seven days after GnRH administration, all the females were slaughtered, their ovaries were collected and then their corporal lutea (CL) was dissected.
Histological Processing
After dissection, four CL from each group were fixed in 10% (v/v) formaldehyde in a phosphate buffer solu-tion of pH 7.2 (PBS) for 24 h and submitted to histolog-ical processing. We made cuts of 6 mm in microtome (Leica RM 2125 RT, Wetzlar, Germany) and stained the sections with periodic acid-Schiff (PAS) (Sigma). This substance has been used to identify blood vessels because it reacts strongly with carbohydrates of the microvascular basal membrane, thereby facilitating the measurement of microvessel density (Ferreira-Dias et al., 2006).
Vascular Density
For assessment of vascular density, we obtained four histologic sections of CL from each animal (a total of 16 sections per group). We used a Nikon DXM1200 dig-ital camera attached to an Olympus BX41 microscope with 4003 magnification to obtain eight photomicro-graphs of distinct randomly selected regions from each section (a total of 128 micrographs per group). For quantification, a cycloid arcs type test system consist-ing of 35 arches and 70 test points was superimposed onto the images as previously described by Gaytan et al. (1999) with some modifications. Each arch had a total length (l/p) of 22 cm, field analysis was delimited by a test area (AT) of 636 cm2and constituted by
inclu-sion (upper and right edges) and excluinclu-sion (lower left margins) lines. We only counted the blood vessels (named profiles in the stereological method) located within the test area and did not touch the exclusion lines. Volume density (Vv) is the ratio of the CL volume occupied by blood vessels: Vv 5 RP/RPCL, where
RP 5 the number of test points which touch the vessels and RPCL5 the total number of test points on the CL.
In these assessments, all vessels in the luteal tissue were also assessed without distinguishing their nature (arterioles, venules, and capillaries).
Scanning Electron Microscopy and Morphometric
The effect of eCG on the microvasculature was assessed by comparing the capillary plexus of two superovulated CL and another of two animals in the control group. To do this, the ovaries of four animals (n 5 2 control and n 5 2 superovulated group) had the ovarian artery cannulated along with its pedicle for the Mercox resin injection (CL-2R, Okenshoji, Tokyo, Japan). To complete polymerization of the resin, the samples were maintained at 608C for 3–4 h. Then, the corrosion of extravascular elements were performed by means of immersion in a 30% sodium hydroxide aque-ous solution for 20 h in a warm oven at 378C. We then soaked the dry molds in distilled water for 24 h to remove residual tissue, s and we glued them onto alu-minum supports (stubs) for embedding in gold and
used a LEO 435 VP Scanning Electron Microscope for visualization. For differentiating arteries and veins, the criteria proposed by Macchiarelli (1995) were adopted regarding the negative impression orientation of the nuclei of endothelial cells: narrow/thin nuclei oriented lengthwise corresponding to arteries, and
randomly-oriented rounded nuclei representing the veins. Button-shaped projections in the capillaries were considered indicative of angiogenesis and partially-filled capillaries representative of degenera-tion (Macchiarelli, 1995; Jiang et al., 2004). The num-bers of angiogenic and degenerative structures were defined per area as previously described by Jiang et al. (2003) with some modifications. In all, eight different microscopic fields of the capillar plexus from each CL were evaluated. The diameter of ten capillaries was measured in the same microscopic fields. To perform these counts and measure capillary diameter, we used Image-Pro PlusVR
software, version 4.5.0.29 for Windows.
Statistical Analysis
We analyzed the vascular density for the normality of residuals using the Anderson–Darling test, trans-formed into base 10 logarithm when necessary and submitted to one-way ANOVA followed by Tukey or Kruskal–Wallis test. Student’s T test was used to com-pare differences in capillary diameter and the number of angiogenic and degenerative structures of the capil-lar plexus in control and superovulated group. P val-ues P 0.05 were considered to determine differences between means. The statistical analyses were per-formed using the GraphPad Prism software (version 4.00 for Windows; GraphPad Prism Software, San Diego, CA).
RESULTS
Microscopy and Vascular Density
The reaction of PAS with the endothelium allowed for the visualization of blood vessels with a circular profile, as well as those which have irregular morphol-ogy in the luteal tissue sections. Figure 1 shows the vessels with irregular morphology, in the intercellular spaces in animals stimulated with eCG. In superovu-lated CL, vessels with large diameter were observed in these intercellular spaces.
The vascular volume density (Vv) increased signifi-cantly in the stimulated and superovulated cows when compared to control (P 0.0001). Nevertheless, vascu-lar density was not different in the stimulated com-pared to the superovulated group (Fig. 2).
Fig. 1. Endothelium stained with PAS in the CL treated with eCG (B stimulated group and C superovulated group) and Control (A). Some blood vessels with a circular profile (arrow) and others with irregular morphology (head arrow) in stimulated CL. C shows vessels with large diameter in these intercellular spaces.
Fig. 2. Vascular volume density of the bovine CL treated with eCG (400 IU in the stimulated group, and 2000 IU in the superovulated group).a-bindicates statistical difference (P 0.0001).
Microvasculature
The CL presented irrigation derived from ovarian cortical arteries and drainage directed to ovarian corti-cal veins. In the control group, a primary luteal artery was observed showing a spiral arrangement derived from the ovarian arterial component, which is evident
on the surface of the CL (Fig. 3a). The angio-architecture of the CLs accompanied the lobular struc-ture of the gland, particularly in animals treated with eCG whose CL showed pronounced interlobular septa and a dense capillary network (Figs. 3a and 3b).
The luteal vascular plexus showed to be highly developed, comprised of numerous typical sinusoidal Fig. 3. (A, C, and E) Control CL and (B, D, and F) superovulated
CL. (A) and (B) Vascular arrangement accompanies the lobular struc-ture of the gland, a characteristic more evident in the superovulated CL (B) with more pronounced interlobular septa (!). In (A) primary luteal artery (capsular artery) spiral on the surface (*) and secondary artery (septal artery) accompanying interlobular septum (!). (C) and
(D) typical sinusoidal tortuous capillary network and small avascular area indicated by #. In the CL control (C), there was a less dense cap-illary network than in superovulated CL (D). In E, numerous degen-erative capillaries are indicated by an arrow. While in F, budding and sprouting capillaries are indicated by an asterisk in superovulated CL.
capillaries in the superovulated CL and control group (Figs. 3c–3d), although the diameter of these capilla-ries in the control group ranged between 8 and 15mm, whereas in the superovulated CL it was 18–35mm. We also observed numerous figures indicative of sprouting angiogenesis, especially in CL treated with eCG (Fig. 3f), whereas many degenerating capillaries in the CL control group (Fig. 3e) were found (Table 1).
DISCUSSION
The present study evaluated vascular changes of CL in cows for the first time, subsequent to i.m. adminis-tration of eCG in doses commonly used in stimulatory and superovulatory treatments of cattle. Based on the results of the density and vascular arrangement obtained by stereology and scanning electron micros-copy, respectively, we found that the use of eCG was able to induce increased angiogenic activity in bovine CL. Other studies have demonstrated that gonadotro-pin also increased the formation of blood vessels in ovarian follicles of mice (Sato et al., 1982) and rats (Koos and LeMaire, 1983). In more recent studies, this eCG angiogenic effect was related to the ability to reg-ulate VEGF expression in granulosa cells, which induced proliferation of endothelial and perivascular cells by a paracrine mechanism, allowing for the for-mation of new blood vessels in the follicular wall (Bar-boni et al., 2000; Jiang et al., 2004). Moreover, other research that assessed the effects of FSH in vitro buf-falo cells showed results of increased expression of VEGF and FGF2 by granulosa cells (Fatima et al, 2013b). This increased microvasculature of CL treated with eCG in the present study may be explained by similar mechanisms, such as other gonadotropins with FSH and hCG also induced an increase in the levels of VEGF in luteal cells (Christenson and Stouffer, 1997; Laitinen et al., 1997; Wulff et al., 2001; Fatima et al., 2013b).
The use of eCG has resulted in improved gestational success for fixed time embryo transfer, independent of the protocol employed for synchronization (Bo et al., 2011). These satisfactory results can be associated with higher progesterone plasma (P4) found in these animals (Soumano et al., 1998). P4 is essential to ensure an adequate uterine environment for growth, implantation, placentation and development of the fetus to term (Spencer et al., 2008). This increase in the synthesis of P4 has been explained by the greater volume of CL changes in the number of large luteal cells (Ferreira et al., 2013; Rostami et al., 2011), and more recently the changes in the number of spherical mitochondria, as well as mitochondria volume
pro-moted by the eCG (Rigoglio et al., 2013). There is still no consensus in the literature on the dose of eCG to more efficiently increase follicular and luteal size, and to produce an earlier rise of P4 serum concentration, thereby improving pregnancy rate. The most fre-quently used dose with present treatment regimens of eCG in TAI protocols for dairy cows in South America is 400 IU (Ferreira et al., 2013). The present work demonstrated that gonadotropin, besides the afore-mentioned effects, also promoted an increase in vascu-lar density; essential to ensure greater blood supply in the CL, responsible for providing hormonal precursors to luteal cells and proper drainage of the produced pro-gesterone (Redmer and Reynolds, 1996). No significant difference in this parameter was observed between the stimulated and superovulated CLs. The stimulatory protocol was sufficient to cause a luteal vasculature improvement critical to the functional performance of the gland, however, in superovulatory dose there was no increase in vascular density of the CL. Miyamoto et al. (2010) demonstrated that the use of eCG for the induction of ovulation in mice repeatedly caused oxida-tive stress in ovarian cells, with a decrease in the amounts of mitochondrial DNA and in the expression of antioxidant enzymes superoxide dismutase 1 and 2 (SOD1 and SOD2). Subsequently, our research group has demonstrated that the eCG superovulatory dose interferes with the structure and function of the cell luteal and their mitochondria (Rigoglio et al., 2013). In the future, there is a need to investigate the possibility of the occurrence of oxidative stress in CL of animals exposed to superovulatory treatment with eCG and its effect on angiogenesis.
In this study, SEM evaluations allowed for identify-ing differences in the vascular plexus of the CL. In superovulated CL, the capillary network had more dilated vessels and was marked by several button-shaped projections in the capillaries. That sprouting of capillaries from preexisting vessels was considered an indicative figure of angiogenesis in previous studies (Jiang et al., 2003; Macchiarelli et al., 2006, 2010; Mar-telli et al., 2009). These angiogenic figures and more dilated capillaries have also been described by Mac-chiarelli (1995) in a CL SEM study of rabbits treated with hCG. More recently, similar characteristics were found in the CL of rats with hypothyroidism and treated with a combination of thyroxine, hCG, and eCG. These morphological findings were correlated with increased expression of VEGF mRNA and bFGF in luteal tissue of the treated group (Macchiarelli et al., 2013). The active angiogenesis has also been described by Jiang et al. (2004) in vascular molds of pigs’ ovarian follicles treated with eCG. The treatment resulted in an atretic follicle population reduction and increased the amount of VEGF in the follicular fluid, so the angiogenesis is also involved in the selection and follicle growth under the regulation of gonadotro-pin. In a superovulated model of immature mice, Guo et al. (2012) demonstrated that Ang-2 expression was strongly induced by eCG 48h post-injection. Miyabaya-shi et al., (2005) also reported a significantly increased mRNA expression of Ang-2, VEGF 120, VEGF 164, Neuropilin-1 and Flt-1 in CL of rats treated with 10 IU of eCG to promote follicular development, followed 48 h later by i.p. administration of 20 IU of hCG. TABLE 1. Capillary diameter and number of angiogenic or
degenerative structures per area of capillary plexus of CL in cows Groups
Control Superovulated Capillary diameter (mm) 11.10 6 1.91a 25.52 6 5.05a Number of angiogenic
structures (number per mm2) 2.6 6 1.2
a 22.2 6 6.7a
Number of degenerative
structures (number per mm2) 6.3 6 2.9 5.4 6 2.2
However, the bovine luteal tissue, treated with eCG, was not increase in mRNA expression of VEGF and FGF2 assessed by quantitative real-time PCR (Fatima et al., 2012). Therefore, this angiogenic effect of eCG in bovine CL needs to be further investigated.
In addition to the blood supply, microvascular per-meability also influences the exchange of substances between cells and the ovarian vascular compartment (Mitsube et al., 2013). In vascular molds analyzed by SEM, we observed that capillaries were more dilated in CL treated with eCG, and a similar effect was also demonstrated with the use of hCG in the CL of rabbits (Macchiarelli, 1995). This action of eCG must also be associated with an increased expression of angiogene-sis factors, as Ang-2 and VEGF are known for their ability to induce increased vascular permeability (Rei-singer et al., 2007; Tuuminen et al., 2014).
The morphometric analysis of vascular casts showed a slight reduction in the number of capillaries in the degeneration in treated CL. This result may indicate that eCG also influences the apoptosis of endothelial cells in the CL of cows. These cells are the first to undergo programmed cell death (PCD) during luteoly-sis (Friedman et al., 2000). There are reports showing that endothelial cells from bovine CL express tumor necrosis factor (TNFa) and its type 1 and type 2 (TNFR1 and TNFR2) receptors, and the decline of pro-gesterone preceding the structural luteolysis is a pre-requisite for apoptosis initiation in these cells induced by TNFa via TNFRI (Friedman et al., 2000; Petroff et al., 1999; Zhao et al., 1998). According to Peluffo et al. (2009), LH decreases the expression of members of the TNFR family in CL mediated by high levels of progestins, and therefore reduces the PCD of the endo-thelial cells. Therefore, our morphological findings indicate that eCG may act through a similar mecha-nism of inhibition of capillary degeneration, but its effect on these cytokines needs to be confirmed by molecular assays.
In summary, the treatment with eCG increased the number of figures indicative of angiogenesis and vascular density in cows’ CL. These morphological findings are evidence of an angiogenic effect of gonado-tropin in the luteal parenchyma.
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