{"paper_id":"dbc1d99f-2e46-461b-b1e7-d4222d203f2c","body_text":"Puberty is the first estrus period in female animals during which ovulation\noccurs. Puberty is related to the hypothalamus–pituitary–gonadal axis\nregulation and the effect of environmental and genetic factors on the\ncoordinating functions of luteinizing and follicle-stimulating hormones\n(Meeran et al., 2003; Redmond et al., 2011; Wankowska et al., 2008; Pool\net al., 2020; Rosa and Bryant, 2003). Puberty is affected by various\nfactors, including genetic mechanisms, nutrient levels, and light duration,\nall of which affect the timing of puberty (Greives et al., 2007; Suttie et\nal., 1985). Studies have shown that the  Lin28B  gene expression in the hypothalamus\nplays an important role in puberty initiation in mammals (Tommiska et\nal., 2010).\nLin28B is a highly conserved RNA-binding protein first discovered in\n Caenorhabditis elegans , and this heterochronic gene regulates nematode development from the larval to adult stages (Ambros, 1989).  Lin28B  was first cloned in human\nhepatocellular carcinoma. It is located on chromosome 6. Lin28B has a very long\n3 \n ′ \n -UTR (untranslated region) and a complementary site for let-7 microRNAs (Guo et al.,\n2006).\nMicroRNAs (miRNAs), a type of endogenous non-coding small RNA composed of\n20–30 nucleotides, are involved in cell development, proliferation,\ndifferentiation, and apoptosis by targeting specific mRNAs, mediating\ntranslational repression, or degrading mRNAs (Baek et al., 2008; Bartel,\n2004; Hwang and Mendell, 2006).  Lin28B  inhibits the biogenesis of various miRNAs,\nincluding the let-7 family miRNAs. Let-7 miRNAs regulate genes related to cell growth\nand differentiation (Peng et al., 2011). Among them, let-7b inhibits cyclin\nD1 expression, which regulates the self-renewal of embryonic stem cells and the\nproliferation and tumorigenicity of cancer cells (Schultz et al., 2008;\nXu et al., 2009; F. Yu et al., 2007).  Lin28B  suppresses miRNA  let-7b  expression to promote\nCD44 \n + \n / Lin28B \n \n + \n  human pancreatic cancer stem cell proliferation and\ninvasion (Shao et al., 2015).\nIn mammals, granulosa cell (GC) proliferation plays an important regulatory\nrole in determining follicle fate and maturation (Douville and Sirard,\n2014; Khan et al., 2016; Saatcioglu et al., 2016). GCs produce estradiol\n(E2), which supports their survival and proliferation and promotes follicle\nmaturation (Chou and Chen, 2018). Mammalian follicle development is a key\nprocess within the ovary, to which GCs directly contribute through their\nproliferation and growth (Lv et al., 2019). In this context, previous\nreports indicate interactions among E2,  Lin28B , and the let-7 family; 17- \n β \n -estradiol and  let-7a – Lin28B  axes synergistically affect the occurrence and development\nof adenomyosis (Huang et al., 2021). Additionally, treating MCF-7 cells\nwith 17- \n β \n -estradiol (E2) resulted in rapid and specifically reduced\n let-7g  expression (Qian et al., 2011).\nThus far, relatively few studies have reported on  Lin28B  and ovine puberty.\nIn the present study, an overexpression vector was constructed from the\nDolang sheep  Lin28B  sequence and transfected into sheep ovarian granulosa cells.\nThis sequence was overexpressed for identifying its effects on  let-7b ,  let-7g , cell\nproliferation, and estrogen secretion. Thus, this study lays a foundation\nfor a better understanding of how  Lin28B  may contribute to the initiation of\npuberty in sheep.\n\nOvaries of Dolang sheep (approximately 3.5 months old) were collected\nimmediately after slaughter and placed in normal saline at 37  \n ∘ \n C\nand brought back to the laboratory for processing. The cumulus–oocyte\ncomplex (COC) was aspirated from follicles with a diameter of 3–8 mm. GCs\nwere collected after serial pipetting, and the follicular fluid and medium\nwere mixed and injected into a sterile 15 mL centrifuge tube. The\nsupernatant was discarded after centrifugation at 175  \n g \n  for 5 min.\nSubsequently, 3 mL of DMEM (Dulbecco's Modified Eagle Medium) was added, stirred gently to mix, and centrifuged at\n175  \n g \n  for 5 min, and the supernatant was discarded. A freshly prepared complete\nmedium (89 % DMEM  \n + \n  10 % fetal bovine serum  \n + \n  1 %\npenicillin  \n / \n  streptomycin) (Gibco, USA) was added, mixed, and transferred to\nPetri dishes. Isolated ovarian granulosa cells were cultured at 37  \n ∘ \n C under 5 % CO \n 2 \n  for subsequent experiments.\nCell slides were placed into 24-well cell culture plates (2  \n × \n  10 \n 4 \n  cells per well), and 1 mL of culture medium was added. The cells were\nincubated for 24 h in an incubator. After the cells had attached to the\nslides, the medium was aspirated, and the cells were washed once with PBS (phosphate-buffered saline)\n(Gibco, USA), fixed with 4 % paraformaldehyde (Solarbio, Beijing, China)\nfor 30 min at 4  \n ∘ \n C, washed three times with PBS for 5 min each,\nand blocked with a blocking solution (0.5 % Triton X-100 mixed with PBS\n1 : 1, plus 10 % goat serum) (Solarbio, Beijing, China) at room temperature\nfor 2 h. The cell slides were incubated with a primary antibody (FSHR\nantibody : PBS  \n = \n  1 : 100) (22665-1-AP, Proteintech, Wuhan, China) at\n4  \n ∘ \n C for 24 h. Subsequently, they were incubated with Goat\nanti-Rabbit IgG (H \n + \n L) Cross-Adsorbed (IgG : PBS  \n = \n  1 : 500) (SA00006-3,\nProteintech, Wuhan, China) for 2 h at room temperature in the dark and\nwashed three times with PBS for 5 min each. The cells were stained with DAPI\n(DAPI : PBS  \n = \n  1 : 1000) (Solarbio, Beijing, China) for 5 min and washed three\ntimes with PBS for 5 min each. One drop of Fluoromount-G (SouthernBiotech,\nUSA) was dropped on the slide, and the side with cells was covered. Images\nwere acquired using a fluorescence microscope (DS-Ri2; Nikon, Japan) and\nexamined with a  \n 100 × \n  objective.\nThe pEGFP-N1 vector was digested with NheI and EcoRI. The 754 bp coding\nsequence of  Lin28B  was amplified from the cDNA of Dolang sheep using qRT-PCR (Takara, Dalian, China) (Xing et al., 2019a). The amplified cDNA\nwas digested with NheI and EcoRI and ligated into the pEGFP-N1 vector to\ngenerate pEGFP-N1– Lin28B , which was verified by DNA sequencing.\nIdentification of sheep granulosa cells (GCs).  (a)  The red marker\nindicates cells expressing FSHR.  (b)  The blue marker indicates DAPI-stained\nnuclei.  (c)  Merge is a red fluorescently labeled FSHR with a blue\nfluorescently DAPI overlay. Bar: 100  \n µ \n m.\nTransfection 24 h cell fluorescence map.  (a)  Fluorescence detection\ngraph of empty-vector group 24 h after transfection.  (b)  Fluorescence\ndetection graph of overexpression of  Lin28B  groups 24 h after transfection. Bar:\n400  \n µ \n m.\nCultured Dolang sheep ovary granulosa cells were suspended with PBS for\nlater use. Trypan blue staining solution (0.4 %) was added to the cell\nsuspension at a cell dye ratio of 1 : 1 ( \n v / v \n ), and the cells were counted\nusing a hemocytometer. According to the manufacturer's instructions for\nLipofectamine 3000,  \n 1 × 10 6 \n  ovarian granulosa cells were plated\nin six-well plates and cultured for 24 h. The cells were transfected using a\nplasmid : transfection reagent ratio of 1 : 3. Cell transfection was divided\ninto three periods: 24 h (0–24 h), 48 h (0–48 h), and 72 h (0–72 h). The\ncells were grouped as target gene, empty vector, and untreated cells. These\nexperiments were run in triplicate. The medium from each time period was\ncollected for measuring estrogen secretion, and cell RNA was extracted by\nTRIzol–chloroform extraction for quantitative real-time PCR (qRT-PCR).\nThe total cell protein was extracted using a kit (TransGen Biotech, China),\nand the protein concentration was detected using a BCA kit (TransGen\nBiotech). A 10 % separating gel and 5 % stacking gel were prepared for\nelectrophoresis. The band was excised and transferred to the membrane,\nfollowing which it was incubated overnight at 4  \n ∘ \n C with the\nprimary antibodies, namely anti-Lin28B (1 : 1000, Abcam, UK) and anti-ACTB\n(1 : 5000, Proteintech). The membrane was then incubated with an\nenzyme-conjugated secondary antibody for 2 h at 37  \n ∘ \n C. The bands\nwere detected using an ELC luminescence kit (Beyotime, Jiangsu, China).\nThe expression of  Lin28B ,  let-7b , and  let-7g  in ovarian granulosa cells 24, 48, and 72 h\nafter transfection was assessed using qRT-PCR.\nFor  Lin28B  mRNA expression, ACTB was selected as the reference gene for normalizing\nmRNA levels. The primer sequences for  Lin28B  and ACTB mRNA are presented in Table 1.\nPrimers used for qRT-PCR analysis.\nFor  let-7b  and  let-7g  quantification, cDNA was synthesized using a miRNA RT Kit (Takara,\nDalian, China). The mature sequences of  let-7b  and  let-7g  were obtained using miRBase\n( https://www.mirbase.org/ , last access: 18 April 2022) and were used to design the primers. The forward\nprimers for  let-7b  and  let-7g  were designed, whereas the reverse primers were included in\nthe Takara miRNA RT Kit. U6 was selected as a reference gene. Table 1 presents\nthe primer sequences used for  let-7b ,  let-7g , and U6 miRNA.\nThe qRT-PCR was performed using the Mir-X miRNA qRT-PCR TB Green Kit (Takara,\nDalian, China). All experiments were run on an Eppendorf device (Eppendorf,\nGermany). Each sample and assay were run in triplicate. The levels of  Lin28B ,\n let-7b , and  let-7g  were calculated using the 2 \n - Δ Δ CT \n  method.\nCell proliferation was measured using a CCK-8 (Cell Counting Kit-8) assay (Beyotime, Jiangsu,\nChina). Cells ( \n 5 × 10 3 \n ) were resuspended in complete DMEM and\nseeded into a 96-well culture plate. The plate was incubated at 37  \n ∘ \n C for 24 h. The incubator was maintained in a humidified\natmosphere of 5 % CO \n 2 \n . WST-8 cell proliferation reagent was added, and\nthe plate was incubated for an additional 2 h. Absorbance at 450 nm was\nmeasured using a microplate reader (BioTek, USA) to analyze the effect of\n Lin28B  overexpression on cell proliferation.\nELISA (Jining, Shanghai, China) was used for detecting E2 concentration in\nthe cell culture media collected at 24, 48, and 72 h.\nSPSS 26.0 software (IBM, Chicago, IL, USA) was used for statistical analysis.\nAll experiments were repeated at least three times, and the experimental results are\npresented as the mean  \n ± \n  standard error. A  \n t \n  test was used for\ncomparing two groups, and a one-way ANOVA was used for testing the differences\namong multiple groups. Statistical significance was set at  \n P < 0.05 \n .\nChanges in  Lin28B  mRNA expression at 24, 48, and 72 h after\ntransfection.  \n * * \n   \n P \n   \n < \n  0.01.\nChanges in Lin28B protein expression at 24, 48, and 72 h after\ntransfection.  \n * * \n   \n P \n   \n < \n  0.01.  (a)  Western blot strip diagram;  (b)  relative expression of Lin28B protein. Notes: (1) 24 h cell group; (2) 24 h empty-vector group; (3) 24 h  Lin28B \noverexpression group; (4) 48 h cell group; (5) 48 h empty-vector group; (6) 48 h  Lin28B  overexpression group; (7) 72 h cell group; (8) 72 h empty-vector group; (9) 72 h  Lin28B  overexpression group.\n\nThrough cell immunofluorescence identification (Fig. 1), this test showed\nthat FSHR was expressed in ovarian granulosa cells, as indicated by red\nimmunofluorescence; DAPI staining appeared as blue (Hong et al., 2022;\nWang et al., 2022). Upon merging these images, the blue fluorescence of DAPI\nand the red fluorescence of FSHR completely overlapped, indicating that the\ncells are of high purity, meeting the requirements of subsequent\nexperiments.\nEffect of  Lin28B  overexpression on the expression of let-7b and let-7g.  \n * * \n   \n P \n   \n < \n  0.01.  (a)  Expression of  let-7b  after transfection with  Lin28B ;  (b)  expression of  let-7g  after\ntransfection with  Lin28B .\nEffect of  Lin28B  overexpression on cell proliferation.  \n * \n   \n P \n   \n < \n  0.05.\nEffect of  Lin28B  overexpression on estradiol.  \n * * \n   \n P \n   \n < \n  0.01.\nAfter transferring the  Lin28B  vector into ovarian granulosa cells, the fluorescence\nbrightness of the test and control groups was examined at 24 h (Fig. 2), and\n Lin28B  mRNA and protein expression levels were measured at 24, 48, and 72 h. After\ntransfection, the mRNA (Fig. 3) and protein expression (Fig. 4) levels of the\n Lin28B  gene in the overexpression group were significantly higher than those in the\nempty-vector group, indicating that the transfection was successful.\nAfter cell transfection, the relative expression levels of  let-7b  and  let-7g  miRNA were\nmeasured using qRT-PCR (Fig. 5). The mRNA expression of the transfected\n Lin28B  group was significantly higher than that of the empty-vector group ( \n P < 0.01 \n ), indicating that  Lin28B  was successfully overexpressed in the\novarian granulosa cells. After the cells were transfected, the expression\nlevels of  let-7b  and  let-7g  in the empty-vector group were significantly higher than those\nin the overexpression group ( \n P < 0.01 \n ), which indicated that\n Lin28B  overexpression significantly inhibited the expression of these miRNAs.\nGranulosa cell proliferation was measured with and without  Lin28B  overexpression.\nThe overexpression of  Lin28B  significantly increased ovarian granulosa cell\nproliferation compared with the empty-vector group ( \n P < 0.05 \n ; Fig. 6).\nFollowing  Lin28B  overexpression, estradiol secretion by granulosa cells was\nmeasured using ELISA. The cell culture medium from cells overexpressing  Lin28B  for 24,\n48, and 72 h was collected. The detection of estradiol is shown in Fig. 7.\nThe concentrations at 24, 48, and 72 h in this group were significantly\nhigher than those in the empty-vector group ( \n P < 0.05 \n ).  Lin28B \noverexpression stably increased estradiol secretion at 24, 48, and 72 h.\nEstradiol levels in these periods were relatively stable, and the difference\nwas not significant ( \n P > 0.05 \n ).\n\nPuberty is the period when the reproductive ability of animals begins, and\nthe length of puberty is related to the reproductive ability of animals\n(Xing et al., 2019b). Animals that reach puberty early\nhave higher reproductive capacity and more offspring. Previous studies\ncorrelate puberty and  Lin28B  mRNA expression in livestock (Cao et al., 2020).\nTo the best of our knowledge, this is the first study to investigate the\nrole of  Lin28B  in sheep GCs. In the present study, an overexpression vector for\n Lin28B  was constructed and transfected into GCs. The transfection was successful,\nas indicated by fluorescence imaging and relative mRNA expression. Following\n Lin28B  overexpression, the  let-7b  and  let-7g  levels were found to be significantly lower than\nthose of the control group.  Let-7b  and  let-7g  expression further decreased with increases\nin  Lin28B  expression. This result is consistent with  Lin28B  mRNA and  let-7b  miRNA expression in\nthe hypothalamus of rats and female rhesus monkeys (Sangiao-Alvarellos et\nal., 2013).\nAdditionally, the present study showed that  Lin28B  overexpression promoted GC\nproliferation and E2 secretion. Other studies have shown similar results,\neven in cancer (Molenaar et al., 2012). According to related studies, the\n Lin28B –let-7 family axis regulates oncogenic cell function and the differentiation,\ngrowth, and metabolism of embryonic stem cells (Murray et al., 2013;\nShyh-Chang and Daley, 2013). Aberrant expression of this axis is frequently\nobserved in severe cancers, such as cervical, bladder, and lung cancers\n(Deng et al., 2017; Guo et al., 2017; Qi et al., 2018; Wu et al., 2019),\nand it normally regulates proliferative and metastatic functions.\nFurthermore, high  Lin28B  expression can promote the proliferation of human\nembryonic stem cells and accelerate their reprogramming process through\nfaster cell division (Hanna et al., 2009; J. Yu et al., 2007). Let-7 miRNAs\nmaintain differentiation patterns and normal development. Let-7 miRNAs and\nestrogen receptor (ER) \n α \n  expressions are correlated (Sun et al.,\n2013). Estrogen secretion is suppressed by let-7 upregulation and decreases in\nER \n α \n  expression (Sun et al., 2016). The present study showed that\n Lin28B  overexpression decreased  let-7b  and  let-7g  levels, promoted GC proliferation, and\nincreased E2 secretion. Considering previous studies and our own, the\n Lin28B –let-7 family axis can be speculated to regulate GC proliferation and E2\nsecretion. However, the mechanism underlying this regulation should be\ninvestigated further.\n\nIn the present study, a vector overexpressing  Lin28B  was transfected into ovarian\ngranulosa cells, whereby  let-7b  and  let-7g  miRNA levels decreased significantly.\nConcurrently,  Lin28B  overexpression can promote proliferation and estrogen\nsecretion in sheep GCs. These findings can be used for further elucidating\nthe regulatory mechanism of  Lin28B  in initiating sheep puberty.","source_license":"CC-BY-4.0","license_restricted":false}