Effects of Acute Inflammation Induced by Lipopolysaccharide (LPS) and Pam3csk4 (TLR2/1 Agonist) on Anti-Müllerian Hormone (AMH) Expression and Serum Levels in the Mouse Ovary

Öz

This study aimed to investigate the effects of acute inflammation induced by lipopolysaccharide (LPS) and Pam3CSK4 (TLR2/1 agonist) during the follicular phase on ovarian and serum anti-Müllerian hormone (AMH) responses in mice. A total of 30 female BALB/c mice aged 6-8 weeks were divided into three groups: control (n = 10), Pam3CSK4 (n = 10), and lipopolysaccharide (LPS) (n = 10). To standardize follicular activity, all mice were initially administered 5 IU equine chorionic gonadotropin (eCG) intraperitoneally (IP). Twenty-four hours following this administration, TLR2/TLR1 and TLR4 activation in the experimental groups was achieved by intraperitoneal administration of 100 µg Pam3CSK4 and 100 µg LPS, respectively, while the control group received an equal volume (100 µL) of phosphate-buffered saline (PBS). Twenty-four hours after the applications, ovarian tissues were collected under general anaesthesia for immunohistochemical evaluation; simultaneously obtained serum samples were analysed for AMH concentrations (ng/mL) using the enzyme immunoassay (EIA) method. Evaluation of the results revealed a marked difference in responses among pathogen-associated molecular patterns (PAMPs). Following administration of the TLR4 activator LPS (6.11 ± 1.3 ng/mL), serum AMH levels were found to be significantly decreased compared to the control group (8.83 ± 1.04 ng/mL) (P<0.05). In contrast, administration of the TLR2/1 agonist Pam3CSK4 did not result in a significant change in serum AMH concentrations (P>0.05). Immunohistochemical analyses qualitatively demonstrated that neither PAMP treatment affected the follicular distribution or cellular localization of AMH in the ovary. In conclusion, LPS altered serum AMH levels, whereas neither PAMP changed the qualitative ovarian localization of AMH within the 24-hour observation period.

Anahtar Kelimeler

• AMH
• LPS
• Ovary
• Pam3CSK4
• Toll-like receptors

Destekleyen Kurum

Erciyes University Scientific Research Projects Coordination Unit

Proje Numarası

TAPD-2025-15362 ve TAPD-2025-15359.

Etik Beyan

All animal experiments were planned based on the reporting and implementation principles defined in the ARRIVE guidelines and were conducted in compliance with Directive 2010/63/EU of the European Union on the protection of animals used for scientific purposes. In addition, all experimental procedures were previously reviewed and approved by the Erciyes University Experimental Animals Ethics Committee (Kayseri, Türkiye) (Ethical Approval No: 20/135).

Teşekkür

The authors would like to thank Assoc. Prof. Dr. Aykut Gram for performing the immunohistochemical analyses and the Erciyes University Scientific Research Projects Coordination Unit for supporting this study.

Lipopolisakkarit (LPS) ve Pam3csk4 (TlR2/1 Agonisti) Tarafından İndüklenen Akut İnflamasyonun Fare Ovaryumunda Anti-Müllerian Hormon (Amh) Ekspresyonu ve Serum Düzeyleri Üzerindeki Etkileri

Öz

Bu çalışma, foliküler fazda lipopolisakkarit (LPS) ve Pam3CSK4 (TLR2/1 agonisti) tarafından indüklenen akut inflamasyonun farelerdeki ovaryum ve serum anti-Müllerian hormon (AMH) yanıtları üzerindeki etkilerini araştırmayı amaçlamıştır. Toplam 30 dişi BALB/c fare (6-8 haftalık) üç gruba ayrıldı: kontrol (n = 10), Pam3CSK4 (n = 10) ve lipopolisakkarit (LPS) (n = 10). Foliküler aktiviteyi standardize etmek için, tüm farelere başlangıçta intraperitoneal (IP) olarak 5 IU at koryonik gonadotropini (eCG) uygulandı. Bu uygulamadan yirmi dört saat sonra, deney gruplarında TLR2/TLR1 ve TLR4 aktivasyonu, sırasıyla 100 µg Pam3CSK4 ve 100 µg LPS'nin intraperitoneal olarak uygulanmasıyla sağlandı; kontrol grubuna ise eşit hacimde (100 µL) fosfat tamponlu salin (PBS) verildi. Uygulamalardan yirmi dört saat sonra, genel anestezi altında ovaryum dokuları immünohistokimyasal değerlendirme için toplandı; eş zamanlı olarak elde edilen serum örnekleri, enzim immün testi (EIA) yöntemi kullanılarak AMH konsantrasyonları (ng/mL) açısından analiz edildi. Sonuçların değerlendirilmesi, patojenle ilişkili moleküler paternler (PAMP'ler) arasında yanıtlarda belirgin bir farklılık olduğunu ortaya koydu. TLR4 aktivatörü LPS'nin (6,11 ± 1,3 ng/mL) uygulanmasının ardından, serum AMH düzeylerinin kontrol grubuna (8,83 ± 1,04 ng/mL) kıyasla anlamlı derecede azaldığı bulundu (P<0,05). Buna karşılık, TLR2/1 agonisti Pam3CSK4'ün uygulanması serum AMH konsantrasyonlarında anlamlı bir değişikliğe yol açmadı (P>0,05). İmmünohistokimyasal analizler, PAMP tedavilerinin hiçbirinin ovaryumda AMH'nin foliküler dağılımını veya hücresel lokalizasyonunu etkilemediğini niteliksel olarak gösterdi. Sonuç olarak, LPS serum AMH seviyelerini değiştirirken, PAMP'lerin hiçbiri 24 saatlik gözlem süresi içinde AMH'nin yumurtalıktaki niteliksel lokalizasyonunu değiştirmemiştir.

Anahtar Kelimeler

• AMH
• LPS
• Ovaryum
• Pam3CSK4
• toll benzeri reseptörler

Destekleyen Kurum

Erciyes Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi

Proje Numarası

TAPD-2025-15362 ve TAPD-2025-15359.

Etik Beyan

Tüm hayvan deneyleri, ARRIVE kılavuzlarında tanımlanan raporlama ve uygulama ilkelerine göre planlanmış ve bilimsel amaçlarla kullanılan hayvanların korunmasına ilişkin Avrupa Birliği Direktifi 2010/63/AB'ye uygun olarak yürütülmüştür. Ayrıca, tüm deneysel prosedürler daha önce Erciyes Üniversitesi Deneysel Hayvanlar Etik Kurulu (Kayseri, Türkiye) tarafından incelenmiş ve onaylanmıştır (Etik Onay No: 20/135).

Teşekkür

Yazarlar, immünohistokimyasal analizleri gerçekleştiren Doç. Dr. Aykut Gram'a ve bu çalışmayı destekleyen Erciyes Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi'ne teşekkürlerini sunarlar.

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