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Simple Summary: Reproductive loss is a major constraint to efficient swine production. Our goal is to better understand the biology of reproduction in female swine. Gonadotropin-releasing hormone II and its receptor are proteins produced by the pig ovary, but their function is not fully understood. To further explore their role in female reproduction, we measured reproductive hormones in the circulating blood of genetically modified pigs that produce less gonadotropin-releasing hormone II receptor throughout the body, and their littermate control siblings during the estrous (reproductive) cycle. Additionally, we compared the morphological characteristics of the ovary and hormone production by the Corpora lutea (ovarian structures that produce the critical pregnancy hormone, progesterone). Ovaries from females with reduced gonadotropin-releasing hormone II receptor levels ovulated 17% fewer oocytes (eggs), contained Corpora lutea with smaller steroidogenic cells, containing 23% less progesterone, with 18% less progesterone into circulation compared with littermate controls. These data suggest that GnRH-II and its receptor play a role in regulating the number of oocytes ovulated, Corpora lutea development, and progesterone production in pigs. A better understanding of female reproduction will lead to novel interventions to enhance the reproductive efficiency of pigs, increasing the productivity and sustainability of the swine industry. Reproduction is classically controlled by gonadotropin-releasing hormone (GnRH-I) and its receptor (GnRHR-I) within the brain. In pigs, a second form (GnRH-II) and its specific receptor (GnRHR-II) are also produced, with greater abundance in peripheral vs. central reproductive tissues. The binding of GnRH-II to GnRHR-II has been implicated in the autocrine/paracrine regulation of gonadal steroidogenesis rather than gonadotropin secretion. Blood samples were collected from transgenic gilts, with the ubiquitous knockdown of GnRHR-II (GnRHR-II KD; n = 8) and littermate controls (n = 7) at the onset of estrus (follicular) and 10 days later (luteal); serum concentrations of 16 steroid hormones were quantified by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Upon euthanasia, ovarian weight (OWT), ovulation rate (OR), and the weight of each excised Corpus luteum (CLWT) were recorded; HPLC-MS/MS was performed on CL homogenates. During the luteal phase, serum progesterone concentration was reduced by 18% in GnRHR-II KD versus control gilts (p = 0.0329). Age and weight at puberty, estrous cycle length, and OWT were similar between lines (p > 0.05). Interestingly, OR was reduced (p = 0.0123), and total CLWT tended to be reduced (p = 0.0958) in GnRHR-II KD compared with control females. Luteal cells in CL sections from GnRHR-II KD gilts were hypotrophic (p < 0.0001). Therefore, GnRH-II and its receptor may help regulate OR, CL development, and progesterone production in gilts. [ABSTRACT FROM AUTHOR]

Due to the nearly exclusive use of artificial insemination, a typical U.S. boar sires ~8,398 progeny per year which is a 99.7% greater gene transmission rate compared with a sow (~23.1 progeny/year). Therefore, the boar has the greatest cumulative impact on swine reproductive performance and drives genetic progress in the herd. However, boar fertility is threatened by climate change. While boar studs are cooled to prevent heat stress, it is cost prohibitive to efficiently cool the six million pregnant sows that are exposed annually, leading to in utero heat stress (IUHS) of developing progeny. The IUHS of the boar reduces the number of pre-spermatogonia in neonatal testes and decimates subsequent semen quality by reducing both total sperm count (24% decrease) and increasing the proportion of abnormal sperm (42% increase). These data suggest that IUHS programs a lifetime of testis dysfunction reducing the breeding potential of boars. However, the biological mechanisms mediating this effect are undefined. The objective of this study was to determine how IUHS affects Leydig cell development and the testicular proteome. Pregnant sows were exposed to either heat stress (28 to 38°C; n = 18) or thermal neutral (17 to 22 °C; n = 18) conditions from day 27 to 57 of gestation. Neonatal boars exposed to IUHS (n = 23) or in utero thermal neutral (IUTN; n = 29) conditions were castrated at 3 days of age. Testes were preserved for histological analysis and proteomics. Leydig cells within IUHS testes were hypertrophic compared with IUTN controls (147.3 ± 1.6 μm² versus 132.8 ± 1.4 μm²; P < 0.0001). Moreover, IUHS decreased the number of Leydig cells (Per microscopy field) compared with IUTN males (72.7 ± 2.0 Leydig cells/field versus 93.5 ± 1.8 Leydig cells/field). The testicular proteome was interrogated in a subset of animals (n = 8/treatment). A total of 519 proteins (32% of those detected) were differentially abundant between IUHS and IUTN testes (278 proteins down-regulated, 241 proteins up-regulated) including proteins important for Sertoli (e.g., insulin-like growth factor binding protein), Leydig (e.g., CYP19), and germ (e.g., glyceraldehyde 3-phosphate dehydrogenase) cell function. Notably, gene ontology analysis revealed that both steroid & cholesterol biosynthesis pathways were enriched (P < 0.0001) indicating differential expression of proteins needed for testicular steroidogenesis. Notably, CYP19 (enzyme expressed in porcine Leydig cells that converts testosterone to 17ß-estradiol) was down-regulated in IUHS testes. Therefore, these data suggest that IUHS leads to Leydig cell dysfunction in boars. The importance of Leydig cells to male reproduction is unequivocal; thus, IUHS represents a mounting threat to male fertility, especially in the face of climate change. [ABSTRACT FROM AUTHOR]

Pigs are the only livestock species encoding functional proteins for both the second form of gonadotropin-releasing hormone (GnRH-II) and its receptor (GnRHR-II). Previously, we determined that GnRHR-II localizes to the connecting piece of ejaculated sperm, whereas GnRH-II is present within seminal plasma, suggesting an autocrine/paracrine interaction influencing porcine sperm function. To examine the GnRH-II/GnRHR-II system further, we produced a swine line with reduced endogenous expression of GnRHR-II (GnRHR-II KD). Therefore, our objective was to assess sperm quality of ejaculates collected from GnRHR-II KD (n = 3) and littermate control (n = 3) boars. Semen was extended (37.5 x 106 sperm/mL) in Androstar Plus and subjected to flow cytometry (Kuster Research and Consulting, Inc.; Atkinson, IL). Assays measured DNA compaction, membrane fluidity and viability, acrosome integrity, mitochondrial depolarization, and morphology. Data were analyzed using the MIXED procedure of SAS; the model included ejaculate as the experimental unit and line as a fixed effect. DNA compaction, membrane fluidity, acrosome integrity, mitochondrial depolarization, and morphology were not statistically different between lines (P > 0.05). However, the percentage of sperm with a viable (intact) plasma membrane was reduced in GnRHR-II KD boars (87.4 ± 0.8%) compared with controls (89.6 ± 0.8%; P < 0.05). Although differences did not reach significance, DNA compaction (a measure of sperm chromatin integrity when undergoing acid stress) was doubled and mitochondrial depolarization (reflective of mitochondrial function) was 11% greater in sperm from transgenic males relative to littermate controls, indicating that GnRHR-II KD sperm are more susceptible to environmental stressors and have altered energy production. Thus, these initial data suggest disruptions in the function and fertilizing capacity of GnRHR-II KD sperm, implying that GnRHR-II may represent a potential biomarker for boar fertility. Supported by USDA/NIFA AFRI (2017-67015-26508) and Hatch Multistate (NEB-26-244) funds. USDA is an equal opportunity provider and employer. [ABSTRACT FROM AUTHOR]

Pigs are the only livestock species encoding functional proteins for both the second form of gonadotropin-releasing hormone (GnRH-II) and its receptor (GnRHR-II), which are uniquely expressed in reproductive and non-reproductive tissues. To examine the physiological role of the GnRH-II/GnRHR-II system, we produced a swine line with reduced endogenous levels of GnRHR-II (GnRHR-II KD); males exhibit 70% diminished testicular GnRHR-II mRNA levels and 82% reduced circulating testosterone concentrations. Given that testosterone impacts metabolism, blood was collected from GnRHR-II KD (n = 5) and littermate control (n = 5) boars via indwelling jugular catheters, with serum isolated and subjected to veterinary diagnostic panels for metabolic analyte examination (PhysLab, Lincoln, NE). Statistical analyses utilized the MIXED procedure of SAS; the model included line as fixed and litter as random effects. Creatine kinase and blood urea nitrogen (BUN):creatinine ratios were elevated, creatinine was reduced (P < 0.01), and thyroxine tended to be decreased (P < 0.10) in GnRHR-II KD compared with control boars. Glucose, BUN, amylase, and lipase levels were not different. Liver products differed in transgenic versus control boars; levels of lactic dehydrogenase, aspartate and alanine aminotransferases (AST; ALT), and gamma-glutamyl transpeptidase were higher, whereas AST:ALT ratios, total protein, albumin, and globulin levels were lower (P < 0.05) in GnRHR-II KD boars. Albumin:globulin ratios and bilirubin (total and direct) did not differ. Additionally, serum cholesterol was decreased (P < 0.05), non-high density lipoproteins (HDLs) and low density lipoproteins (LDLs) tended to be decreased (P < 0.10), and triglycerides, HDLs, and cholesterol:HDL ratios did not differ between GnRHR-II KD and control males. These data suggest metabolic disruption in GnRHR-II KD boars, which may be due to suppressed gonadal steroidogenesis or ubiquitous knockdown of GnRHR-II expression. Supported by USDA/NIFA AFRI (2017-67015-26508) and Hatch Multistate (NEB-26-244) funds. USDA is an equal opportunity provider and employer. [ABSTRACT FROM AUTHOR]

Kisspeptin is a major regulator of gonadotropin secretion in pigs. Previously, CRISPR/Cas9 knockout of KISS1 was used to develop a mosaic parental line of pigs to generate offspring that would not need castration due to loss of kisspeptin. The current goal was to characterize growth and reproductive development of F1 pigs from this parental line. Body weights, gonadotropin concentrations and gonadal development were measured from birth through development (boars to 220 d of age, n = 42; gilts to 160 d of age, n = 36). Testosterone, skatole, and androstenone were also measured in boars. Blood samples were collected by jugular venipuncture for quantification of serum hormones, gonadal tissues collected for gross morphology and histology, and a fat biopsy collected (boars) for skatole and androstenone analysis. Body weight did not differ with genotype. There were no differences between KISS1+/+ and heterozygote KISS1+/- animals for most parameters measured. Gonadotropin concentrations were reduced in KISS1-/- boars and gilts compared with KISS1+/+ and KISS1+/- animals (P < 0.05). Concentrations of testosterone in serum and both androstenone and skatole in adipose were less in KISS1-/- boars than in KISS1+/+ and KISS1+/- boars (P < 0.05). Hypogonadism was in all KISS1-/- gilts and boars. These data indicate that knocking out KISS1 causes hypogonadotropic hypogonadism but does not negatively affect growth in pigs. Only one KISS1 allele is needed for normal gonadotropin secretion and gonadal development, and accumulation of compounds in adipose leading to boar taint., (Published by Oxford University Press on behalf of Society for the Study of Reproduction 2024.)

Since genetic engineering of pigs can benefit both biomedicine and agriculture, selecting a suitable gene promoter is critically important. The cytomegalovirus (CMV) promoter, which can robustly drive ubiquitous transgene expression, is commonly used at present, yet recent reports suggest tissue-specific activity in the pig. The objective of this study was to quantify ZsGreen1 protein (in lieu of CMV promoter activity) in tissues from pigs harboring a CMV-ZsGreen1 transgene with a single integration site. Tissue samples ( n =35) were collected from neonatal hemizygous ( n =3) and homozygous ( n =3) piglets and ZsGreen1 abundance was determined via immunoblotting. ZsGreen1 was detected in all tissues, except hypothalamus, kidney cortex and oviduct. The expression patterns of homozygous and hemizygous piglets were similar ( P >0.05). However, quantification revealed that ZsGreen1 protein levels were tissue-specific. Within neural/endocrine tissues, ZsGreen1 abundance was highest in the anterior pituitary gland, intermediate in the cerebellum and lowest in the cerebrum, spinal cord and posterior pituitary ( P <0.05). In the digestive system, ZsGreen1 was more abundant in the salivary gland than esophagus, stomach, pancreas, duodenum, jejunum, ileum, spleen, colon, gallbladder and liver ( P <0.05). Interestingly, ZsGreen1 amounts also differed within an organ ( i.e. , the right ventricle had 3-fold higher levels than the other heart chambers; P <0.05). These results provide useful information for the use of the CMV promoter to drive transgene expression in the pig. Moreover, this swine model represents a novel resource of ZsGreen1-labeled organs and a valuable tool to advance genome editing research.