Bone morphogenetic proteins (BMPs) are among the subfamily members of the transforming growth factor and are regarded as multifunctional growth factors. BMPs have functional roles in embryonic development, including cartilage, neural development, and postnatal bone formation (10). Besides their function in organs, BMPs function in different biological events in the ovary such as folliculogenesis, ovulation, and steroidogenesis (11, 12). BMPs compose the extensive part of the transforming growth factor (TGFβ) superfamily members. Some of the member of BMPs (BMP-2, - 3, -4, -6, -7) which forms heterodimer initiate BMP signaling through receptors of BMP; type I (BMP-IA and BMPR-IB) and type II (BMPR-II). Once ligand interaction implemented, signal transduction is accomplished via phosphorylation of type I receptor by type II receptor.

Recently, during spontaneous regression, some of BMPs and their own receptors have been shown to be expressed in the human corpus luteum and are adversely modulated by the human chorionic gonadotropin (13). Contrary to the widespread role in folliculogenesis, limited number of studies (13-15) have focused on the modulation and function of BMPs throughout luteinization and luteolysis. Accordingly, we aimed to assess the expression patterns of BMP genes in the induced luteolysis of ovine CL.

Gene Expression: We evaluated BMPs mRNA expression levels with the aid of qPCR with specific primers of BMP genes (Table 1). We prepared qPCR mix as follows: 10 μl qPCR master mix (Luna® Universal Protocol, #M3003, NEB), 2 μL cDNA, 2 μL primer (10 pMol), and 6 μL water to final volume of 20 μL ¹⁸. PCR conditions were arranged as previously defined ¹⁹. We ran melting analysis as follows: 95°C for 2 min, the fluorescence signal was captured at 1°C increment starting from 55°C up to 95°C (Bio-Rad CFX96; Bio-Rad, USA). To normalize the gene expression data, SDHA was employed as a housekeeping gene for gene expression analysis ²⁰. Positive and negative control runs were included to eliminate genomic contamination. We performed each gene duplicate.

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Table 1: BMP gene primers for qPCR

Statistical Analysis: qPCR (Ct) data were used to calculate relative expression ¹⁸. The method of 2−ΔΔCt was employed to calculate relative expression ²¹. qPCR normalized data were analyzed by t-test and ANOVA with Tukey’s post hoc test. P<0.05 value was set as significance.

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Figure 1: Expression of BMP2 mRNA in C12, PG4, and PG16. Data was presented as mean±standard error of means (SEM)

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Figure 2: Expression of BMP4 mRNA in C12, PG4, and PG16. **: P<0.01. Data was presented as mean±standard error of means (SEM)

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Figure 3: Expression of BMP6 mRNA in C12, PG4, and PG16. *: P<0.05 and **: P<0.01. Data was presented as mean±standard error of means (SEM)

BMPR1A mRNA steady-state levels were demonstrated in C12 and induced luteolysis of PG4, and PG16 in Figure 4. Expression of BMPR1A mRNA was found to be greater in PG16 than in PG4 (P<0.05). However, the level of BMPR1A mRNA did not change between induced luteolysis groups (PG4 and PG16) and C12 (P>0.05). BMPR1B mRNA steady-state levels were demonstrated in C12 and induced luteolysis of PG4, and PG16 in Figure 5. While expression of BMPR1B mRNA was greater in PG16 than in PG4 and C12 (P<0.05), it was shown to be lower in PG4 than in C12 (P<0.05). BMPR2 mRNA steady-state levels were demonstrated in C12 and induced luteolysis of PG4, and PG16 in Figure 6. Although BMPR2 mRNA was found to be lower in PG16 than in PG4 and C12 (P<0.05), this was not significant (P>0.05). Expression of BMPR2 mRNA did not change between C12 and PG4 (P>0.05).

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Figure 4: Expression of BMPR1A mRNA in C12, PG4, and PG16. *: P<0.05. Data was presented as mean±standard error of means (SEM)

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Figure 5: Expression of BMPR1B mRNA in C12, PG4, and PG16. *: P<0.05 and ****: P<0.0001. Data was presented as mean±standard error of means (SEM)

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Figure 6: Expression of BMPR2 mRNA in C12, PG4, and PG16. Data was presented as mean±standard error of means (SEM)

Throughout the oestrous cycle, luteal regression in ruminants and other species is induced by the release of PGF2α, which reaches the CL from the uterus. Luteolysis is prevented when the embryo is present and emerges in cyclic animals ^21,22. Endogenous or exogenous administration of PGF2α facilitates a signalling of events causing to irreversible death of CL. In the entire process, CL undergoes significant changes in terms of its steroidogenic capacity, vascularization, extracellular matrix regeneration and cell viability ^{23, 24}.

In recent years, many functional genomic studies have elucidated the underlying mechanism of PGF2α over luteolysis ^{25, 26}. Studies reveal that factors produced by uterine or exogenous PGF2α mediate a variety of processes from decreased steroid production to apoptotic cell death. Factors such as bone morphogenic proteins (BMP), tumor necrosis factor-alpha (TNFα), and activin A may have inhibitory effects on StAR expression ^27-30.

It has been reported that some BMPs cause luteolysis by suppressing StAR expression and P4 generation in women and granulosa cells ^{13, 31}. In our study, this may be consistent with the regulation of BMP genes that we showed BMP6 mRNA were upregulated in PG16 compared to C12. Also, we previously reported that StAR mRNA was shown to be sharply decreased in PG16, thus explaining BMPs’ inhibitory effects on StAR as identified previously in humans CL ¹³. BMP2 and BMP4 mRNA expression was demonstrated to be increased in induced luteolysis of cattle CL after 12 hours after PGF2α treatment, but BMP6 was after 2 hours ³². BMP2 abundance was reported to link/be linked with diminished P4 in cattle CL ³³. However, we have not observed any changes in the expression of BMP2 and BMP4 mRNA levels in our study. BMPRs assessed in the current study were expressed in the ovine CL during luteolysis. We have not detected significant regulation for BMPR2 as in induced luteolysis of bovine CL ³². However, BMPR1A mRNA were not regulated in induced luteolysis against C12, whereas BMPR1B mRNA was upregulated in PG16. In line with our study, in rats, the greater levels of BMPR1B was reported in CL regression which implies its involvement in luteolysis ¹⁴.

In conclusion, considering mRNA expression of BMP2, BMP4, BMP6, BMPR1A, BMPR1B, and BMPR2, BMPs appeared to have functional role in corpus luteum. We may suggest that expression patterns of some BMP genes regulate luteolysis of ovine CL.

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